��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Human Disease-causing Mutations Disrupt an N-C-terminal Interaction and Channel Function of Bestrophin 1

Mutations in the human bestrophin 1 (hBest1) chloride channel cause Best vitelliform macular dystrophy. Although mutations in its transmembrane domains were found to alter biophysical properties of the channel, the mechanism for disease-causing mutations in its N and C termini remains elusive. We hypothesized that these mutations lead to channel dysfunction through disruption of an N-C-terminal interaction. Here, we present data demonstrating that hBest1 N and C termini indeed interact both in vivo and in vitro. In addition, using a spectrum-based fluorescence resonance energy transfer method, we showed that functional hBest1 channels in the plasma membrane were multimers. Disease-causing mutations in the N terminus (R19C, R25C, and K30C) and the C terminus (G299E, D301N, and D312N) caused channel dysfunction and disruption of the N-C interaction. Consistent with the functional and biochemical results, mutants D301N and D312N clearly reduced fluorescence resonance energy transfer signal, indicating that the N-C interaction was indeed perturbed. These results suggest that hBest1 functions as a multimer in the plasma membrane, and disruption of the N-C interaction by mutations leads to hBest1 channel dysfunction.Extensive evidence has shown that bestrophins are anion channels when expressed heterologously in cell lines (1–9) and represent a type of endogenous calcium-activated chlorine channel in several cell types (10–12). Mutations disrupting hBest1 channel function lead to Best vitelliform macular dystrophy (Best disease) (13). Bestrophin 1 channels have been reported to exist as oligomers (2, 14). Results from immunoprecipitation experiments suggested that hBest1 exists as tetramers or pentamers when expressed heterologously (2), whereas a hydrodynamic study based on the calculations of the molecular mass of the Best1-detergent complex concluded that native porcine Best1 channels are dimers (14). Besides the uncertainty about the subunit stoichiometry, it is still unclear how bestrophin subunits assemble and interact in functional channels.Understanding how disease-causing mutations might adversely affect the assembly and interaction of bestrophin subunits is important for elucidating the molecular mechanism underlying the Best disease. The hBest1 protein contains six transmembrane domains (TMD1–6) with intracellularly located N and C termini. There are three “hot spots” of disease-causing mutations in hBest1: the N-terminal region, TMD2, and the C terminus proximal to TMD6 (13, 15). Multiple lines of evidence support that mutations in TMD2 alter the biophysical properties of the channel (3, 4, 7). How mutations distributed around the N- and C-terminal regions cause Best disease is less clear, although strong evidence recently suggested that certain mutations in the C terminus disrupt hBest1 channel gating by Ca²⁺ (16). N-C-terminal interaction of multimeric channels has been demonstrated to be involved in the activation of many channel types including inward rectifier K⁺ channels (17) and cyclic nucleotide-gated channels (18–20). In the present study we tested the hypothesis that an interaction between the N and C termini plays an important role in normal hBest1 channel function, and weakening or disruption of this interaction by mutations leads to channel dysfunction.To test our hypothesis, we introduced disease-causing mutations into the N- or C-terminal regions of hBest1 expressed in HEK293 cells and tested the effects of mutations on subunit interaction and channel function with a combination of electrophysiological, biochemical, and optical methods. We found that these mutations not only disrupted channel function but also caused N- and C-terminal dissociation both in vitro and in vivo. The findings suggest a novel molecular mechanism for the mutations in hBest1 to cause human Best disease.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Identification of Protein Domains That Control Proton and Calcium Sensitivity of ASIC1a

The acid-sensing ion channels (ASICs) open in response to extracellular acidic pH, and individual subunits display differential sensitivity to protons and calcium. ASIC1a acts as a high affinity proton sensor, whereas ASIC2a requires substantially greater proton concentrations to activate. Using chimeras composed of ASIC1a and ASIC2a, we determined that two regions of the extracellular domain (residues 87–197 and 323–431) specify the high affinity proton response of ASIC1a. These two regions appear to undergo intersubunit interactions within the multimeric channel to specify proton sensitivity. Single amino acid mutations revealed that amino acids around Asp³⁵⁷ play a prominent role in determining the pH dose response of ASIC1a. Within the same region, mutation F352L abolished PcTx1 modulation of ASIC1a. Surprisingly, we determined that another area of the extracellular domain was required for calcium-dependent regulation of ASIC1a activation, and this region functioned independently of high affinity proton sensing. These results indicate that specific regions play overlapping roles in pH-dependent gating and PcTx1-dependent modulation of ASIC1a activity, whereas a distinct region determines the calcium dependence of ASIC1a activation.The acid-sensing ion channels (ASICs)³ are proton-gated ion channels expressed in neurons throughout the central and peripheral nervous system (1–3). ASICs are activated by extracellular acidosis, and protons act as ligands triggering channel opening (4). Disruption of the accn2 gene (which encodes ASIC1) dramatically reduces proton-gated currents in central neurons and alters a variety of behaviors, including fear, learning, and memory (5, 6). ASIC1 also contributes to neuronal damage and death during the prolonged acidosis accompanying cerebral ischemia (7). Specifically, mice with disruptions in the accn2 gene display 60% smaller lesion size compared with normal mice in models of stroke (8). Application of PcTx1, a venom peptide that prevents ASIC1a activation, is similarly neuroprotective, even when administered hours after injury (8, 9). Thus, ASIC1a represents a novel pharmacological target for the prevention of neuronal death following stroke.Mammals have four genes encoding ASICs (accn1 to -4) that encode at least six different ASIC subunits (1–3, 10). Like all members of the DEG/ENaC family, individual ASIC subunits have two transmembrane regions separated by a large cysteine-rich extracellular region. Three ASIC subunits associate to form homomeric or heteromeric channels with distinct biophysical characteristics (11–14). Specifically, ASIC1a homomeric channels activate at pH values much closer to neutral pH compared with ASIC2a homomeric channels. The high affinity proton sensitivity of ASIC1a plays a prominent role in acidosis-induced neuronal death, and modulators that alter the pH dose response of ASIC1a affect neuronal sensitivity to prolonged acidosis (8, 9, 15). For example, the neuroprotective venom peptide PcTx1 increases the proton sensitivity of the ASIC1a channel, allowing the channel to desensitize at neutral pH and become unresponsive to subsequent acidic shifts in pH (16, 17). The large extracellular region of ASIC1a is thought to be the site of proton/modulator interaction and governs the characteristics of channel gating (10, 11, 18). However, the exact molecular mechanisms defining ASIC1a activation and the protein domains that are responsible for the apparent proton sensitivity of ASIC1a remain unclear. Here, we used chimeras containing specific regions from both ASIC1a and ASIC2a to identify the specific protein regions that confer high affinity proton sensing, PcTx1 sensitivity, and calcium modulation to ASIC1a.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Ligand-specific Conformational Changes in the ��1 Glycine Receptor Ligand-binding Domain

Understanding the activation mechanism of Cys loop ion channel receptors is key to understanding their physiological and pharmacological properties under normal and pathological conditions. The ligand-binding domains of these receptors comprise inner and outer β-sheets and structural studies indicate that channel opening is accompanied by conformational rearrangements in both β-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on α1 glycine receptors to compare changes mediated by the agonist, glycine, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner β-sheet, we labeled residues in loop 2 and in binding domain loops D and E. At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes in the inner β-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors.Glycine receptor (GlyR)3 chloride channels are pentameric Cys loop receptors that mediate fast synaptic transmission in the nervous system (1, 2). This family also includes nicotinic acetylcholine receptors (nAChRs), γ-aminobutyric acid type A and type C receptors, and serotonin type 3 receptors. Individual subunits comprise a large ligand-binding domain (LBD) and a transmembrane domain consisting of four α-helices (M1–M4). The LBD consists of a 10-strand β-sandwich made of an inner β-sheet with six strands and an outer β-sheet with four strands (3). The ligand-binding site is situated at the interface of adjacent subunits and is formed by loops A–C from one subunit and loops D–F from the neighboring subunit (3).The activation mechanism of Cys loop receptors is currently the subject of intense investigation because it is key to understanding receptor function under normal and pathological conditions (4, 5). Based on structural analysis of Torpedo nAChRs, Unwin and colleagues (6, 7) originally proposed that agonist binding induced the inner β-sheet to rotate, whereas the outer β-sheet tilted slightly upwards with loop C clasping around the agonist. These movements were thought to be transmitted to the transmembrane domain via a differential movement of loop 2 (β1-β2) and loop 7 (β6-β7) (both part of the inner β-sheet) and the pre-M1 domain (which is linked via a β-strand to the loop C in the outer sheet). The idea of large loop C movements accompanying agonist binding is supported by structural and functional data (3, 8–13). However, a direct link between loop C movements and channel gating has proved more difficult to establish. Although computational modeling studies have suggested that this loop may be a major component of the channel opening mechanism (14–18), experimental support for this model is not definitive. Similarly, loop F is also thought to move upon ligand binding, although there is as yet no consensus as to whether these changes represent local or global conformational changes (11, 19–21). Recently, a comparison of crystal structures of bacterial Cys loop receptors in the closed and open states revealed that although both the inner and outer β-sheets exhibit different conformations in closed and open states, the pre-M1 domain remains virtually stationary (22, 23). It is therefore relevant to question whether loop C, loop F, and pre-M1 movements are essential for Cys loop receptor activation.Strychnine is a classical competitive antagonist of GlyRs (24, 25), and to date there is no evidence that it can produce LBD structural changes. In this study we use voltage-clamp fluorometry (VCF) to compare glycine- and strychnine-induced conformational changes in the GlyR loops 2, C, D, E, and F and the pre-M1 domain in an attempt to determine whether they signal ligand-binding events, local conformational changes, or conformational changes associated with receptor activation.In a typical VCF experiment, a domain of interest is labeled with an environmentally sensitive fluorophore, and current and fluorescence are monitored simultaneously during ligand application. VCF is ideally suited for identifying ligand-specific conformational changes because it can report on electrophysiologically silent conformational changes (26), such as those induced by antagonists. Indeed, VCF has recently provided valuable insights into the conformational rearrangements of various Cys loop receptors (19, 21, 27–33).     

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.Oligomeric heat shock protein 27 (Hsp27)² is a ubiquitous mammalian protein with a variety of functions in health and disease (1–8). These functions include ATP-independent chaperone activity in response to environmental stress, e.g. heat shock and oxidative stress, control of apoptosis, and regulation of actin cytoskeleton dynamics. Hsp27 is a member of the α-crystallin small heat shock protein family of which αB-crystallin is the archetype. These proteins are characterized by an α-crystallin domain of 80–90 residues consisting of roughly eight β-strands that form an intermolecular β-sheet interaction interface within a dimer, the basic building subunit of the oligomer (2, 4, 9–11).Hsp27 is in equilibrium between high molecular weight oligomers and much lower molecular weight multimers. It has been reported that unphosphorylated Hsp27 includes predominantly a distribution of high molecular species ranging in size from 12-mer to 35-mer (12–19). Phosphorylation of Hsp27 at serines 15, 78, and 82 by the p38-activated MAPKAP-2 kinase (20–22) or the use of the triple Ser-to-Asp phospho-mimic results in a major shift in the equilibrium toward much smaller multimers (23) and in an alteration of its function (1, 3, 6, 7, 24, 25). The size distribution of the smaller species has been reported to be between monomer and tetramer (12–16, 18, 19).Small heat shock proteins, including Hsp27, behave as ATP-independent molecular chaperones during cellular heat shock. They bind partially unfolded proteins and prevent their aggregation until the proteins can be refolded by larger ATP-dependent chaperones or are digested (7, 8, 26). This function includes the up-regulation and/or phosphorylation of Hsp27.It is not entirely clear what the role of Hsp27 size and phosphorylation state plays in its heat shock function because there are conflicting results in the literature. Some in vitro studies concluded that the unphosphorylated oligomeric Hsp27 (or the murine isoform Hsp25) protects proteins against aggregation better than does the phosphorylation mimic (13, 19, 27), whereas others found no difference (16, 28, 29), and still other studies found that the mimic protects better than does the unphosphorylated wild type (27, 30, 31). In-cell studies found that phosphorylation of Hsp27 was essential for thermo-protection of actin filaments (32), and the Hsp27 phosphorylation mimic decreased inclusion body formation better than did unphosphorylated Hsp27 (33). This study was undertaken to investigate the molecular chaperone function of Hsp27 by correlating chaperone activity with Hsp27 size and by comparing fully phosphorylated Hsp27 with its phospho-mimic.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.     

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.Protein glycosylation is a biologically significant and complex post-translational modification, involved in cell-cell and receptor-ligand interactions (1–4). In fact, clinical biomarkers and therapeutic targets are often glycoproteins (5–9). Comprehensive glycoprotein characterization, involving glycosylation site identification, glycan structure determination, site occupancy, and glycan isoform distribution, is a technical challenge particularly for quantitative profiling of complex protein mixtures (10–13). Both N- and O-glycans are structurally heterogeneous (i.e. a single site may have different glycans attached or be only partially occupied). Therefore, the MS¹ signals from glycopeptides originating from a glycoprotein are often weaker than from non-glycopeptides. In addition, the ionization efficiency of glycopeptides is low compared with that of non-glycopeptides and is often suppressed in the presence of non-glycopeptides (11–13). When the MS signals of glycopeptides are relatively high in simple protein digests then diagnostic sugar oxonium ion fragments produced by, for example, front-end collisional activation can be used to detect them. However, when peptides and glycopeptides co-elute, parent ion scanning is required to selectively detect the glycopeptides (14). This can be problematic in terms of sensitivity, especially for detecting glycopeptides in digests of complex protein extracts.Isolation of glycopeptides from proteolytic digests of complex protein mixtures can greatly enhance the MS signals of glycopeptides using reversed-phase LC-ESI-MS (RPLC-ESI-MS) or MALDI-MS (15–24). Hydrazide chemistry is used to isolate, identify, and quantify N-linked glycopeptides effectively, but this method involves lengthy chemical procedures and does not preserve the glycan moieties thereby losing valuable information on glycan structure and site occupancy (15–17). Capturing glycopeptides with lectins has been widely used, but restricted specificities and unspecific binding are major drawbacks of this method (18–21). Under reversed-phase LC conditions, glycopeptides from tryptic digests of gel-separated glycoproteins have been enriched using graphite powder medium (22). In this case, however, a second digestion with proteinase K is required for trimming down the peptide moieties of tryptic glycopeptides so that the glycopeptides (typically <5 amino acid residues) essentially resemble the glycans with respect to hydrophilicity for subsequent separation. Moreover, the short peptide sequences of the proteinase K digest are often inadequate for de novo sequencing of the glycopeptides.Glycopeptide enrichment under normal-phase LC (NPLC) conditions has been demonstrated using various hydrophilic media and different capture and elution conditions (23–28). NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZIC®-HILIC column (23–27) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28). However, NPLC is neither effective for enriching less hydrophilic glycopeptides, e.g. the five high mannose type glycopeptides modified by 7–11 monosaccharide units from a tryptic digest of ribonuclease b (RNase B), nor for enriching O-linked glycopeptides of bovine fetuin using a ZIC-HILIC column (23). The use of Sepharose medium for enriching glycopeptides yielded only modest recovery of glycopeptides (28). In addition, binding of hydrophilic non-glycopeptides with these hydrophilic media contaminates the enriched glycopeptides (23, 28).We have recently developed an ion-pairing normal-phase LC (IP-NPLC) method to enrich glycopeptides from complex tryptic digests using Sepharose medium and salts or bases as ion-pairing reagents (29). Though reasonably effective the technique still left room for significant improvement. For example, the method demonstrated relatively modest glycopeptide selectivity, providing only 16% recovery for high mannose type glycopeptides (29). Here we report on a new IP-NPLC method using acids as ion-pairing reagents and polyhydroxyethyl aspartamide (A) as the stationary phase for the effective isolation of tryptic glycopeptides. The method was developed and evaluated using a tryptic digest of RNase B and fetuin mixture. In addition, we demonstrate that O-linked glycopeptides can be effectively isolated from a fetuin tryptic digest by IP-NPLC after removal of the N-linked glycans by PNGase F.The new IP-NPLC method was used to enrich N-linked glycopeptides from the tryptic digests of protein extracts of wild-type (wt) and PglD mutant strains of Campylobacter jejuni NCTC 11168. C. jejuni has a unique N-glycosylation system that glycosylates periplasmic and inner membrane proteins containing the extended N-linked sequon, D/E-X-N-X-S/T, where X is any amino acid other than proline (30–32). The N-linked glycan of C. jejuni has been previously determined to be GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3]-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1 (BacGalNAc₅Glc residue mass: 1406 Da), where Bac is 2,4-diacetamido-2,4,6-trideoxyglucopyranose (30). In addition, the glycan structure of C. jejuni is conserved, unlike in eukaryotic systems (30–32). IP-NPLC recovered close to 100% of the bacterial N-linked glycopeptides with virtually no contamination of non-glycopeptides. Furthermore, we demonstrate for the first time that acetylation of bacillosamine is incomplete in the wt using IP-NPLC and label-free MS.