The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.     

Engineered Protein Connectivity to Actin Mimics PDZ-dependent Recycling of G Protein-coupled Receptors but Not Its Regulation by Hrs

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the β2-adrenergic receptor (β2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na⁺/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the δ-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors (GPCRs)² comprise the largest family of transmembrane signaling receptors expressed in animals and transduce a wide variety of physiological and pharmacological information. While these receptors share a common 7-transmembrane-spanning topology, structural differences between individual GPCR family members confer diverse functional and regulatory properties (1-4). A fundamental mechanism of GPCR regulation involves agonist-induced endocytosis of receptors via clathrin-coated pits (4). Regulated endocytosis can have multiple functional consequences, which are determined in part by the specificity with which internalized receptors traffic via divergent downstream membrane pathways (5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed by the δ-opioid receptor (δOR), contributes to proteolytic down-regulation of receptor number and produces a prolonged attenuation of subsequent cellular responsiveness to agonist (8, 9). Trafficking of internalized GPCRs via a rapid recycling pathway, a major route traversed by the β2-adrenergic receptor (β2AR), restores the complement of functional receptors present on the cell surface and promotes rapid recovery of cellular signaling responsiveness (6, 10, 11). When co-expressed in the same cells, the δOR and β2AR are efficiently sorted between these divergent downstream membrane pathways, highlighting the occurrence of specific molecular sorting of GPCRs after endocytosis (12).Recycling of various integral membrane proteins can occur by default, essentially by bulk membrane flow in the absence of lysosomal sorting determinants (13). There is increasing evidence that various GPCRs, such as the β2AR, require distinct cytoplasmic determinants to recycle efficiently (14). In addition to requiring a cytoplasmic sorting determinant, sequence-dependent recycling of the β2AR differs from default recycling in its dependence on an intact actin cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs (hepatocyte growth factor receptor substrate) (11, 14). Compared with the present knowledge regarding protein complexes that mediate sorting of GPCRs to lysosomes (15, 16), however, relatively little is known about the biochemical basis of sequence-directed recycling or its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called PDZbd), and PDZ-mediated protein association(s) with this sequence appear to be primarily responsible for its endocytic sorting activity (17-20). Fusion of this sequence to the cytoplasmic tail of the δOR effectively re-routes endocytic trafficking of engineered receptors from lysosomal to recycling pathways, establishing the sufficiency of the PDZbd to function as a transplantable sorting determinant (18). The β2AR-derived PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ proteins (21, 22). A well-established biochemical function of NHERF/EBP50 family proteins is to associate integral membrane proteins with actin-associated cytoskeletal elements. This is achieved through a series of protein-interaction modules linking NHERF/EBP50 family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to actin filaments (23-26). Such indirect actin connectivity is known to mediate other effects on plasma membrane organization and function (23), however, and NHERF/EBP50 family proteins can bind to additional proteins potentially important for endocytic trafficking of receptors (23, 25). Thus it remains unclear if actin connectivity is itself sufficient to promote sequence-directed recycling of GPCRs and, if so, if such connectivity recapitulates the normal cellular regulation of sequence-dependent recycling. In the present study, we took advantage of the modular nature of protein connectivity proposed to mediate β2AR recycling (24, 26), and extended the opioid receptor fusion strategy used successfully for identifying diverse recycling sequences in GPCRs (27-29), to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can be effectively bypassed by linking receptors to ERM family proteins in the absence of the PDZbd itself. Further, we establish that the protein connectivity network can be further simplified by fusing receptors to an interaction module that binds directly to actin filaments. We found that bypassing the PDZ-mediated interaction using either domain is sufficient to mimic the ability of the PDZbd to promote efficient, actin-dependent recycling of receptors. Hrs-dependent regulation, however, which is characteristic of sequence-dependent recycling of wild-type receptors, was recapitulated only by the fused PDZbd and not by the proposed downstream interaction modules. These results support a relatively simple architecture of protein connectivity that is sufficient to mimic the core recycling activity of the β2AR-derived PDZbd, but not its characteristic cellular regulation. Given that an increasing number of GPCRs have been shown to bind PDZ proteins that typically link directly or indirectly to cytoskeletal elements (17, 27, 30-32), the present results also suggest that actin connectivity may represent a common biochemical principle underlying sequence-dependent recycling of various GPCRs.     

Intracellular Trafficking of Presenilin 1 Is Regulated by ��-Amyloid Precursor Protein and Phospholipase D1

Excessive accumulation of β-amyloid peptides in the brain is a major cause for the pathogenesis of Alzheimer disease. β-Amyloid is derived from β-amyloid precursor protein (APP) through sequential cleavages by β- and γ-secretases, whose enzymatic activities are tightly controlled by subcellular localization. Delineation of how intracellular trafficking of these secretases and APP is regulated is important for understanding Alzheimer disease pathogenesis. Although APP trafficking is regulated by multiple factors including presenilin 1 (PS1), a major component of the γ-secretase complex, and phospholipase D1 (PLD1), a phospholipid-modifying enzyme, regulation of intracellular trafficking of PS1/γ-secretase and β-secretase is less clear. Here we demonstrate that APP can reciprocally regulate PS1 trafficking; APP deficiency results in faster transport of PS1 from the trans-Golgi network to the cell surface and increased steady state levels of PS1 at the cell surface, which can be reversed by restoring APP levels. Restoration of APP in APP-deficient cells also reduces steady state levels of other γ-secretase components (nicastrin, APH-1, and PEN-2) and the cleavage of Notch by PS1/γ-secretase that is more highly correlated with cell surface levels of PS1 than with APP overexpression levels, supporting the notion that Notch is mainly cleaved at the cell surface. In contrast, intracellular trafficking of β-secretase (BACE1) is not regulated by APP. Moreover, we find that PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell surface accumulation of PS1 in an APP-independent manner. Our results clearly elucidate a physiological function of APP in regulating protein trafficking and suggest that intracellular trafficking of PS1/γ-secretase is regulated by multiple factors, including APP and PLD1.An important pathological hallmark of Alzheimer disease (AD)⁴ is the formation of senile plaques in the brains of patients. The major components of those plaques are β-amyloid peptides (Aβ), whose accumulation triggers a cascade of neurodegenerative steps ending in formation of senile plaques and intraneuronal fibrillary tangles with subsequent neuronal loss in susceptible brain regions (1, 2). Aβ is proteolytically derived from the β-amyloid precursor protein (APP) through sequential cleavages by β-secretase (BACE1), a novel membrane-bound aspartyl protease (3, 4), and by γ-secretase, a high molecular weight complex consisting of at least four components: presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and presenilin enhancer-2 (PEN-2) (5, 6). APP is a type I transmembrane protein belonging to a protein family that includes APP-like protein 1 (APLP1) and 2 (APLP2) in mammals (7, 8). Full-length APP is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi apparatus. Most secreted Aβ peptides are generated within the trans-Golgi network (TGN), also the major site of steady state APP in neurons (9–11). APP can be transported to the cell surface in TGN-derived secretory vesicles if not proteolyzed to Aβ or an intermediate metabolite. At the cell surface APP is either cleaved by α-secretase to produce soluble sAPPα (12) or reinternalized for endosomal/lysosomal degradation (13, 14). Aβ may also be generated in endosomal/lysosomal compartments (15, 16). In contrast to neurotoxic Aβ peptides, sAPPα possesses neuroprotective potential (17, 18). Thus, the subcellular distribution of APP and proteases that process it directly affect the ratio of sAPPα to Aβ, making delineation of the mechanisms responsible for regulating trafficking of all of these proteins relevant to AD pathogenesis.Presenilin (PS) is a critical component of the γ-secretase. Of the two mammalian PS gene homologues, PS1 and PS2, PS1 encodes the major form (PS1) in active γ-secretase (19, 20). Nascent PSs undergo endoproteolytic cleavage to generate an amino-terminal fragment (NTF) and a carboxyl-terminal fragment (CTF) to form a functional PS heterodimer (21). Based on observations that PSs possess two highly conserved aspartate residues indispensable for γ-secretase activity and that specific transition state analogue γ-secretase inhibitors bind to PS1 NTF/CTF heterodimers (5, 22), PSs are believed to be the catalytic component of the γ-secretase complex. PS assembles with three other components, NCT, APH-1, and PEN-2, to form the functional γ-secretase (5, 6). Strong evidence suggests that PS1/γ-secretase resides principally in the ER, early Golgi, TGN, endocytic and intermediate compartments, most of which (except the TGN) are not major subcellular sites for APP (23, 24). In addition to generating Aβ and cleaving APP to release the APP intracellular domain, PS1/γ-secretase cleaves other substrates such as Notch (25), cadherin (26), ErbB4 (27), and CD44 (28), releasing their respective intracellular domains. Interestingly, PS1/γ-secretase cleavage of different substrates seems to occur at different subcellular compartments; APP is mainly cleaved at the TGN and early endosome domains, whereas Notch is predominantly cleaved at the cell surface (9, 11, 29). Thus, perturbing intracellular trafficking of PS1/γ-secretase may alter interactions between PS1/γ-secretase and APP, contributing to either abnormal Aβ generation and AD pathogenesis or decreased access of PS1/γ-secretase to APP such that Aβ production is reduced. However, mechanisms regulating PS1/γ-secretase trafficking warrant further investigation.In addition to participating in γ-secretase activity, PS1 regulates intracellular trafficking of several membrane proteins, including other γ-secretase components (nicastrin, APH-1, and PEN-2) and the substrate APP (reviewed in Ref. 30). Intracellular APP trafficking is highly regulated and requires other factors such as mint family members and SorLA (2). Moreover, we recently found that phospholipase D1 (PLD1), a phospholipid-modifying enzyme that regulates membrane trafficking events, can interact with PS1, and can regulate budding of APP-containing vesicles from the TGN and delivery of APP to the cell surface (31, 32). Interestingly, Kamal et al. (33) identified an axonal membrane compartment that contains APP, BACE1, and PS1 and showed that fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I, implying a traffic-regulating role for APP. Increased APP expression is also shown to decrease retrograde axonal transport of nerve growth factor (34). However, whether APP indeed regulates intracellular trafficking of proteins including BACE1 and PS1/γ-secretase requires further validation. In the present study we demonstrate that intracellular trafficking of PS1, as well as that of other γ-secretase components, but not BACE1, is regulated by APP. APP deficiency promotes cell surface delivery of PS1/γ-secretase complex and facilitates PS1/γ-secretase-mediated Notch cleavage. In addition, we find that PLD1 also regulates intracellular trafficking of PS1 through a different mechanism and more potently than APP.     

Sequential protein kinase C (PKC)-dependent and PKC-independent protein kinase D catalytic activation via Gq-coupled receptors: differential regulation of activation loop Ser(744) and Ser(748) phosphorylation

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1–5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸ revealed striking PKC-independent phosphorylation of Ser⁷⁴⁸ as well as Ser⁷⁴⁴ phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20–90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser⁷⁴⁴ and Ser⁷⁴⁸ is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as a fundamental signal transduction mechanism triggered by multiple extracellular stimuli, including hormones, neurotransmitters, chemokines, and growth factors (1). Many of these agonists bind to G protein-coupled receptors (GPCRs),⁴ activate heterotrimeric G proteins and stimulate isoforms of the phospholipase C family, including β, γ, δ, and ε (reviewed in Refs. 1 and 2). Activated phospholipase Cs catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate mobilizes Ca²⁺ from intracellular stores (3, 4) whereas DAG directly activates the classic (α, β, and γ) and novel (δ, ε, η, and θ) isoforms of PKC (5–7). Although it is increasingly recognized that each PKC isozyme has specific functions in vivo (5–8), the mechanisms by which PKC-mediated signals are propagated to critical downstream targets remain incompletely defined.PKD, also known initially as PKCμ (9, 10), and two recently identified serine protein kinases termed PKD2 (11) and PKCν/PKD3 (12, 13), which are similar in overall structure and primary amino acid sequence to PKD (14), constitute a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase group (15) and separate from the previously identified PKCs (14). Salient features of PKD structure include an N-terminal regulatory region containing a tandem repeat of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain) that confers high affinity binding to phorbol esters and DAG (9, 16, 17), followed by a pleckstrin homology (PH) domain that negatively regulates catalytic activity (18, 19). The C-terminal region of the PKDs contains its catalytic domain, which is distantly related to Ca²⁺-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity maintained by the N-terminal domain, which represses the catalytic activity of the enzyme by autoinhibition. Consistent with this model, deletions or single amino acid substitutions in the PH domain result in constitutive kinase activity (18–20). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (21). In response to cellular stimuli, PKD is converted from a low activity form into a persistently active form that is retained during isolation from cells, as shown by in vitro kinase assays performed in the absence of lipid co-activators (21, 22). PKD activation has been demonstrated in response to engagement of specific GPCRs either by regulatory peptides (23–30) or lysophosphatidic acid (27, 31, 32); signaling through G_q, G₁₂, G_i, and Rho (27, 31–34); activation of receptor tyrosine kinases, such as the platelet-derived growth factor receptor (23, 35, 36); cross-linking of B-cell receptor and T-cell receptor in B and T lymphocytes, respectively (37–40); and oxidative stress (41–44).Throughout these studies, multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF 109203X or Gö 6983) that do not directly inhibit PKD catalytic activity (21, 22), implying that PKD activation in intact cells is mediated, directly or indirectly, through PKCs. In line with this conclusion, cotransfection of PKD with active mutant forms of “novel” PKCs (PKCs δ, ε, η, and θ) resulted in robust PKD activation in the absence of cell stimulation (21, 44–46). Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade in response to multiple GPCR agonists in a broad range of cell types, including normal and cancer cells (reviewed in Ref. 14). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as the activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (reviewed in Ref. 14). Collectively, these findings demonstrated the existence of rapidly activated PKC-PKD protein kinase cascade(s) and raised the possibility that some PKC-dependent biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular activities and processes, including signal transduction (30, 47–49), chromatin modification (50), Golgi organization and function (51, 52), c-Jun function (47, 53, 54), NFκB-mediated gene expression (43, 55, 56), and cell survival, migration, and differentiation and DNA synthesis and proliferation (reviewed in Ref. 14). Thus, mounting evidence indicates that PKD has a remarkable diversity of both its signal generation and distribution and its potential for complex regulatory interactions with multiple downstream pathways, leading to multiple responses, including long term cellular events. Despite increasing recognition of its importance, very little is known about the mechanism(s) of sustained PKD activation as opposed to the well documented rapid, PKC-dependent PKD activation.The results presented here demonstrate that prolonged GPCR-induced PKD activation is mediated by sequential PKC-dependent and PKC-independent phases of regulation. We report here, for the first time, that PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies expand previous models of PKD regulation by identifying a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.     

The inhalation anesthetic desflurane induces caspase activation and increases amyloid beta-protein levels under hypoxic conditions

Perioperative factors including hypoxia, hypocapnia, and certain anesthetics have been suggested to contribute to Alzheimer disease (AD) neuropathogenesis. Desflurane is one of the most commonly used inhalation anesthetics. However, the effects of desflurane on AD neuropathogenesis have not been previously determined. Here, we set out to assess the effects of desflurane and hypoxia on caspase activation, amyloid precursor protein (APP) processing, and amyloid β-protein (Aβ) generation in H4 human neuroglioma cells (H4 naïve cells) as well as those overexpressing APP (H4-APP cells). Neither 12% desflurane nor hypoxia (18% O₂) alone affected caspase-3 activation, APP processing, and Aβ generation. However, treatment with a combination of 12% desflurane and hypoxia (18% O₂) (desflurane/hypoxia) for 6 h induced caspase-3 activation, altered APP processing, and increased Aβ generation in H4-APP cells. Desflurane/hypoxia also increased levels of β-site APP-cleaving enzyme in H4-APP cells. In addition, desflurane/hypoxia-induced Aβ generation could be reduced by the broad caspase inhibitor benzyloxycarbonyl-VAD. Finally, the Aβ aggregation inhibitor clioquinol and γ-secretase inhibitor L-685,458 attenuated caspase-3 activation induced by desflurane/hypoxia. In summary, desflurane can induce Aβ production and caspase activation, but only in the presence of hypoxia. Pending in vivo confirmation, these data may have profound implications for anesthesia care in elderly patients, and especially those with AD.An estimated 200 million patients worldwide undergo surgery each year. Several reports have suggested that anesthesia and surgery may facilitate development of Alzheimer disease (AD)⁴ (1–3). A recent study also reported that patients having coronary artery bypass graft surgery under general anesthesia are at increased risk for AD as compared with those having percutaneous transluminal coronary angioplasty under local anesthesia (4).Genetic evidence, confirmed by neuropathological and biochemical findings, indicates that excessive production and/or accumulation of amyloid β-protein (Aβ) play a fundamental role in the pathology of AD (reviewed in Refs. 5 and 6). Aβ is produced via serial proteolysis of amyloid precursor protein (APP) by aspartyl protease β-site APP-cleaving enzyme (BACE), or β-secretase, andγ-secretase. BACE cleaves APP to generate a 99-residue membrane-associated C terminus fragment (APP-C99). APP-C99 is further cleaved by γ-secretase to release 4-kDa Aβ and β-amyloid precursor protein intracellular domain (7–9). Presenilin and γ-secretase co-fractionate as a detergent-sensitive, high molecular weight complex (10) that includes at least three other proteins, nicastrin/APH-2, APH-1, and PEN-2, all of which are necessary and sufficient for γ-secretase activity (11–13). Increasing evidence indicates that apoptosis is associated with a variety of neurodegenerative disorders, including AD (Refs. 14–17; reviewed in Ref. 18). Aβ has been shown to cause caspase activation and apoptosis, which can in turn potentiate Aβ generation (16, 19–28). Finally, fibrillar aggregates of Aβ and oligomeric species of Aβ are more neurotoxic (29–37).Perioperative factors, including hypoxia (38–42), hypocapnia (43), and anesthetics (44–47), have been reported to potentially contribute to AD neuropathogenesis. These perioperative factors may also cause post-operative cognitive dysfunction, a dementia associated with surgery and anesthesia, by triggering AD neuropathogenesis.Isoflurane, sevoflurane, and desflurane are the most commonly used inhalation anesthetics. It has been reported that isoflurane enhances the oligomerization and cytotoxicity of Aβ (44) and induces apoptosis (48–51). Our recent studies have shown that a clinically relevant concentration of isoflurane can lead to caspase-3 activation, decrease cell viability, alter APP processing, and increase Aβ generation in human H4 neuroglioma cells overexpressing human APP (45–47). Loop et al. (49) reported that isoflurane and sevoflurane, but not desflurane, can induce caspase activation and apoptosis in human T lymphocytes. However, effects of desflurane and desflurane plus other perioperative risk factors, e.g. hypoxia, on APP processing and Aβ generation have not been assessed.In the present study, we set out to determine effects of desflurane, hypoxia, and the combination of the two (desflurane/hypoxia) on caspase-3 activation, APP processing, and Aβ generation in H4 human neuroglioma cells (H4 naïve cells) and H4 naïve cells stably transfected to express full-length (FL) APP (H4-APP cells). We also investigated whether the caspase inhibitor, Z-VAD, the γ-secretase inhibitor L-685,458, and the Aβ aggregation inhibitor clioquinol could attenuate desflurane/hypoxia-induced caspase-3 activation and Aβ generation.     

Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers

The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPα and the neurotoxic β-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPα, as sAPPα binding disrupts APP dimers, and this disruption of APP dimers by sAPPα is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPα. These findings suggest a potentially beneficial effect of increasing sAPPα production or disrupting APP dimers for neuronal survival.The amyloid precursor protein (APP)⁴ is known both for its important role in the development and plasticity of the nervous system (1–6) and for its involvement in Alzheimer disease (AD) (7, 8). Despite intensive research efforts, the initial events that lead to the prevalent sporadic, i.e. non-familial, forms of AD are still unclear. Furthermore, although a higher gene dose of APP (9) or the presence of pathological APP mutations is sufficient to induce familial AD (for review, see Ref. 10), the exact pathological mechanism that is triggered by APP is still under debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are thought to contribute to synaptic dysfunction and neurotoxicity (11, 12). On the other hand, the α-secretase-derived extracellular fragment of APP (sAPPα), which is present at lower levels in AD patients than in controls (13), has been shown to be beneficial for memory function, to possess neuroprotective properties, and to counteract the effects of Aβ (14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration in AD (19–24); however, the signal transduction pathway for transmembrane APP remains unknown, although several potential regulatory proteins, glycosaminoglycans, and metal ions are known to bind with high affinity to APP and sAPPα (25, 26). The most common form of signal transduction for single-pass transmembrane proteins is the ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the dimerization of transmembrane APP has been implied several times in the past. Several studies have investigated the effects of presumed dimer-breaking perturbations on biological read-outs, such as the production of Aβ (27, 28), but without directly measuring the APP aggregation state, or have investigated the aggregation state of APP subdomains, often reconstituted in cell-free systems (27–32). Dimerization interfaces in both the extracellular and the transmembrane domain have been suggested.In the studies investigating the aggregation state of full-length APP, most of the employed methods, such as chemical cross-linking and co-immunoprecipitation, do not lend themselves readily to a rigorous quantitative analysis of the abundance of potentially instable dimers (31, 33), whereas in other cases the use of chimeras may have influenced the dimerization potential or precluded the search for a natural stimulus (23, 34). The only previously reported direct observation of APP dimerization by Förster resonance energy transfer (FRET) microscopy uses an assay in which the FRET efficiency varies with the level of overexpression (35). Therefore, a concentration-dependent FRET component due to nonspecific stochastic encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the selective detection of APP dimers on the surface of live cells, where they would encounter ligands, they could not differentiate between subpopulations of APP. This may be one reason why no natural ligand of APP has ever been shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective sAPPα (36–39). The ligand-dependent dimerization of sAPPα in solution (40) and its origination from transmembrane APP suggest that APP might serve as receptor for sAPPα, but this binding has never been experimentally shown.     

Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase C�� to Associate with Microtubules and to Recruit Dynein

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Cι (aPKCι) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCι in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCι were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin, we characterized the distribution of tyrosinated/detyrosinated α-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated α-tubulin; however, aPKCι was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCι-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway and associates with vesicular tubular clusters (VTCs)² located between the endoplasmic reticulum (ER) and the cis-Golgi compartment (1, 2). VTCs are pleomorphic structures that sort anterograde-directed cargo from recycling proteins and trafficking machinery retrieved to the ER (3-6). Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that results in the release of vesicles containing the recycling protein p53/p58 (7). In that regard, we have previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical PKC ι (aPKCι) are Rab2 effectors that interact directly with the Rab2 amino terminus and with each other (8, 9). Their interaction requires Src-dependent tyrosine phosphorylation of GAPDH and aPKCι (10). Moreover, GAPDH is a substrate for aPKCι (11). GAPDH catalytic activity is not required for ER to Golgi transport indicating that GAPDH provides a specific function essential for membrane trafficking from VTCs independent of glycolytic function (9). Indeed, phospho-GAPDH influences MT dynamics in the early secretory pathway (11).GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs) (12) and subsequently was shown to interact with the carboxyl terminus of α-tubulin (13). The binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays or bundles (14, 15). GAPDH has also been reported to possess membrane fusogenic activity, which is inhibited by tubulin (16). Similarly, aPKC associates directly with tubulin and promotes MT stability and MT remodeling at specific intracellular sites (17-21). It may not be coincidental that these two Rab2 effectors influence MT dynamics because recent studies indicate that the cytoskeleton plays a central role in the organization and operation of the secretory pathway (22).MTs are dynamic structures that grow or shrink by the addition or loss of α- and β-tubulin heterodimers from the ends of protofilaments (23). Their assembly and stability is regulated by a variety of proteins traditionally referred to as microtubule-associated proteins (MAPs). In addition to the multiple α/β isoforms that are present in eukaryotes, MTs undergo an assortment of post-translational modifications, including acetylation, glycylation, glutamylation, phosphorylation, palmitoylation, and detyrosination, which further contribute to their biochemical heterogeneity (24, 25). It has been proposed that these tubulin modifications regulate intracellular events by facilitating interaction with MAPs and with other specific effector proteins (24). For example, the reversible addition of tyrosine to the carboxyl terminus of α-tubulin regulates MT interaction with plus-end tracking proteins (+TIPs) containing the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with dynein-dynactin (27-29). Additionally, MT motility and cargo transport rely on the cooperation of the motor proteins kinesin and dynein (30). Kinesin is a plus-end directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor, and therefore the motors transport vesicular cargo toward the opposite end of a MT track (31).Although MT assembly does not appear to be directly regulated by small GTPases, Rab proteins provide a molecular link for vesicle movement along MTs to the appropriate target (22, 32-34). In this study, the potential interaction of Rab2 with MTs and motor proteins was characterized. We found that Rab2 does not bind directly to preassembled MTs but does associate when both GAPDH and aPKCι are present and bound to MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin (Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds MAPs/effector proteins/motors associates with VTCs in response to Rab2. To that end, we determined that Rab2-promoted dynein/dynactin binding to membranes and that the recruitment required aPKCι.     

Polo-like Kinase 2 (PLK2) Phosphorylates ��-Synuclein at Serine 129 in Central Nervous System

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system.The importance of α-synuclein to the pathogenesis of Parkinson disease (PD)⁴ and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1–3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4–6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7–9), which increase levels of α-synuclein with no alteration in sequence, also cause PD or DLB.α-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11, 12). Phosphorylation at tyrosine residues has been observed by some investigators (13, 14) but not by others (10–12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress α-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of α-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate α-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an α-synuclein kinase. The ability of PLK2 to directly phosphorylate α-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates α-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brain support a role for PLK2 as an α-synuclein kinase in vivo.     

Detecting Morphologically Distinct Oligomeric Forms of ��-Synuclein

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.Parkinson disease (PD)² is the second most common neurodegenerative disorder of the elderly, affecting more than 500,000 people in the United States (1), with 50,000 new cases reported each year at an annual cost estimated at 10 billion dollars per year. Pathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and formation of fibrillar cytoplasmic inclusions known as Lewy bodies and Lewy neurites (2, 3). The protein α-synuclein has been strongly linked to PD (4, 5) and other related neurodegenerative disorders (6, 7) by several lines of evidence. 1) It is the major component of the hallmark Lewy body aggregates associated with PD. 2) Mutations (A53T, A30P, and E46K, where A30P is human A30P α-synuclein; A53T is human A53T α-synuclein; E46K is human E46K α-synuclein) or multiplication in the α-synuclein gene have been linked to familial PD (8–10). 3) Overexpression of α-synuclein in transgenic mice and Drosophila has been shown to induce the formation of PD-like pathological phenotypes and behavior, although the animal models do not in general replicate neuronal loss patterns (11, 12).α-Synuclein is a small protein (14 kDa) expressed mainly in brain tissues and is primarily localized at the presynaptic terminals of neurons (13). The primary structure of α-synuclein consists of three distinct regions. The N-terminal region of α-synuclein includes the mutation sites associated with familial PD (A53T, A30P, and E46K) and contains six imperfectly conserved repeats (KTKEGV) that may facilitate protein-protein binding. This repeat section is predicted to form amphipathic α-helices, typical of the lipid-binding domain of apolipoproteins (14). The central region, non-amyloid component, is extremely hydrophobic and includes a 12-residue stretch (VTGVTAVAQKTV) that is essential for aggregation (15). The C-terminal region is enriched with acidic glutamate and aspartate residues and is responsible for the chaperone function of α-synuclein (16).α-Synuclein normally exists as an unfolded protein, but it can adopt several different folded conformations depending on the environment, including small aggregates or oligomers, spherical and linear protofibrils, as well as the fibrillar structure found in Lewy bodies (14, 15). A growing body of evidence implicates the oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death (16–18). Several different oligomeric forms of α-synuclein including spherical, annular (19), pore-like (20), and dopamine-stabilized structures have been identified in vitro (21).α-Synuclein is considered a cytosolic protein, and consequently its pathogenic effect was assumed to be limited to the cytoplasm of single cells. However, recent studies have suggested that α-synuclein also has extracellular pathogenic effects (22–25). α-Synuclein was detected in blood plasma and cerebrospinal fluid in both monomeric and oligomeric forms (22–25), and the presence of significantly elevated levels of oligomeric species of α-synuclein has been reported extracellularly in plasma and cerebrospinal fluid samples from patients with PD (23). Furthermore, various studies have shown that aggregated α-synuclein added extracellularly to the culture medium is cytotoxic (26–32).Despite all these studies, it is still not clear how the various aggregate forms of α-synuclein are involved in the progression of PD. Therefore, reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only for fundamental studies of how α-synuclein aggregates affect cell function but also as potential diagnostic and therapeutic agents for PD.Recently, we reported inhibition of both aggregation and extracellular toxicity of α-synuclein in vitro by a single chain variable domain antibody fragment (scFv) that specifically recognized an oligomeric form of α-synuclein (32). In this study, we describe a second scFv (syn-10H) that binds a larger later stage oligomeric form of α-synuclein than the previously reported scFv. The syn-10H scFv neutralizes α-synuclein-induced toxicity in both undifferentiated and differentiated SH-SY5Y human neuroblastoma cell line and inhibits α-synuclein aggregation in vitro. The syn-10H scFv reacts specifically with homogenized PD brain tissue but does not cross-react with similarly treated samples taken from Alzheimer disease (AD) or healthy brain samples. Such scFvs therefore have potential value as diagnostic reagents to identify the presence of specific oligomeric species in PD tissue and fluid samples. The scFvs also have value as therapeutic agents as they can be used either extracellularly or expressed intracellularly (intrabodies) to prevent formation of toxic aggregates in vivo whether inside or outside of cells. Intrabodies have been used efficiently to neutralize toxic effects of different pathogenic agents, including α-synuclein (33–36). Moreover, immunization studies in mouse models of PD have shown that extracellular antibodies can reduce accumulation of intracellular aggregates of α-synuclein (37), thereby providing precedent for the use of scFvs in potential passive vaccination strategies for treating PD.     

Effects of Differential Glycosylation of Glycodelins on Lymphocyte Survival

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.Glycodelin is a member of the lipocalin family. It consists of 180 amino acid residues (1) with two sites of N-linked glycosylation. There are four reported glycodelin isoforms, namely glycodelin-A (amniotic fluid isoform, GdA),⁴ glycodelin-F (follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S (seminal plasma, GdS) (2–5). Among the four glycodelin isoforms, only the N-glycan structures of GdA and GdS have been previously determined. This was achieved using fast atom bombardment mass spectrometry (6, 7). The glycan structures of GdA and GdS are completely different. In GdA, the Asn-28 site carries high mannose, hybrid, and complex-type structures, whereas the second Asn-63 site is exclusively occupied by complex-type glycans (6). The major non-reducing epitopes characterized in the complex-type glycans are Galβ1–4GlcNAc (lacNAc), GalNAcβ1–4GlcNAc (lacdiNAc), NeuAcα2–6Galβ1–4GlcNAc (sialylated lacNAc), NeuAcα2–6GalNAcβ1–4GlcNAc (sialylated lacdiNAc), Galβ1–4(Fucα1–3)GlcNAc (Lewis-x), and GalNAcβ1–4(Fucα1–3)GlcNAc (lacdiNAc analog of the blood group substance Lewis-x) (6). Many of these oligosaccharides are rare in other human glycoproteins. GdS glycans are unusually fucose-rich, and the major complex type glycan structures are bi-antennary glycans with Lewis-x and Lewis-y antennae. Glycosylation of GdS is highly site-specific. Asn-28 contains only high mannose structures, whereas Asn-63 contains only complex type glycans. More than 80% of the complex glycans have 3–5 fucose residues/glycan, and none of the glycans is sialylated, which is unusual for a secreted human glycoprotein (7). The glycan structures of GdF and GdC are not known, although they differ in lectin-binding properties and isoelectric point from the other two glycodelin isoforms (5).Glycans are involved in various intracellular, intercellular, and cell-matrix recognition events (8, 9). Glycosylation determines the biological activities of the glycodelin isoforms (2, 10). For example, both GdA and GdF inhibit the spermatozoa-zona pellucida binding (11) via fucosyltransferase-5 (12), but only the latter inhibits progesterone-induced acrosome reaction, thus preventing a premature acrosome reaction of the spermatozoa. There is evidence that cumulus cells can convert exogenous GdA and -F to GdC, the physicochemical properties of which suggest that it is differently glycosylated compared with GdA/F (5). Moreover, GdC stimulated spermatozoa-zona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound GdA and -F (4, 5). GdS suppresses capacitation probably via its inhibitory activity on cholesterol efflux from spermatozoa (13).Except for the effects on fertilization, GdA is involved in fetomaternal defense. This glycodelin isoform suppresses proliferation and induces apoptosis of T cells (2) and inhibits natural killer cell (14) and B-cell (15) activities. Glycosylation is involved in the binding of GdA to receptors on T cells (16). The sialic acid of GdA contributes to the apoptotic activity in T cells (17, 18) and binding to CD45, a potential GdA receptor (16). The importance of glycosylation in glycodelin is further shown by the absence of immunosuppressive activities in GdS with different glycosylation (18). The immunomodulating activities of GdF and GdC are unknown.Our previous work showed that glycans are indispensable for the different glycodelins to exhibit their binding activities and biological effects (13, 19, 20). The present study aims to identify the effect of all four glycodelin isoforms on lymphocyte viability, cell death, and interleukin-2 (IL-2) secretion and to correlate these bioactivities with their glycosylation patterns determined by mass spectrometry.     

Structural and Mechanistic Insights into Lunatic Fringe from a Kinetic Analysis of Enzyme Mutants

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of β1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-α-l-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases, including multiple sclerosis (1), several forms of cancer (2-4), cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (5), and spondylocostal dysostosis (SCD)³ (6-8). The transmembrane Notch signaling receptor is activated by members of the DSL (Delta, Serrate, Lag2) family of ligands (9, 10). In the endoplasmic reticulum, O-linked fucose glycans are added to the epidermal growth factor-like (EGF) repeats of the Notch extracellular domain by protein O-fucosyltransferase 1 (11-13). These O-fucose monosaccharides can be elongated in the Golgi apparatus by three highly conserved β1,3-N-acetylglucosaminyltransferases of the Fringe family (Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals) (14-16). The formation of this GlcNAc-β1,3-Fuc-α1, O-serine/threonine disaccharide is necessary and sufficient for subsequent elongation to a tetrasaccharide (15, 19), although elongation past the disaccharide in Drosophila is not yet clear (20, 21). Elongation of O-fucose by Fringe is known to potentiate Notch signaling from Delta ligands and inhibit signaling from Serrate ligands (22). Delta ligands are termed Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on Drosophila Notch can be recapitulated in Notch ligand in vitro binding assays using purified components, suggesting that the elongation of O-fucose by Fringe alters the binding of Notch to its ligands (21). Although Fringe also appears to alter Notch-ligand interactions in mammals, the effects of elongation of the glycan past the O-fucose monosaccharide is more complicated and appears to be cell type-, receptor-, and ligand-dependent (for a recent review see Ref. 23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc disaccharide (14-16). They belong to the GT-A-fold of inverting glycosyltransferases, which includes N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase I (17, 18). The mechanism is presumed to proceed through the abstraction of a proton from the acceptor substrate by a catalytic base (Asp or Glu) in the active site. This creates a nucleophile that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its configuration from α (on the nucleotide sugar) to β (in the product) (24, 25). The enzyme then releases the acceptor substrate modified with a disaccharide and UDP. The Mfng structure (26) leaves little doubt as to the identity of the catalytic residue, which in all likelihood is aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc soaked into the crystals (26) showed density only for the UDP portion of the nucleotide-sugar donor and no density for two loops flanking either side of the active site. The presence of flexible loops that become ordered upon substrate binding is a common observation with glycosyltransferases in the GT-A fold family (18, 25). Density for the entire donor was observed in the structure of rabbit N-acetylglucosaminyltransferase I (27). In this case, ordering of a previously disordered loop upon UDP-GlcNAc binding may have contributed to increased stability of the donor. In the case of bovine β1,4-galactosyltransferase I, a section of flexible random coil from the apo-structure was observed to change its conformation to α-helical upon donor substrate binding (28). Both loops in Lfng are highly conserved, and we have mutated a number of residues in each to test the hypothesis that they interact with the substrates. The mutagenesis strategy was also guided by docking of an EGF-O-fucose acceptor substrate into the active site of the Lfng model as well as comparison of the Lfng model with a homology model of the β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on thrombospondin type 1 repeats (29, 30). The β3GlcT is predicted to be a GT-A fold enzyme related to the Fringe family (17, 18, 29).