Lactobacillus acidophilus Alleviates Platelet-Activating Factor-Induced Inflammatory Responses in Human Intestinal Epithelial Cells

Probiotics have been used as alternative prevention and therapy modalities in intestinal inflammatory disorders including inflammatory bowel diseases (IBD) and necrotizing enterocolitis (NEC). Pathophysiology of IBD and NEC includes the production of diverse lipid mediators, including platelet-activating factor (PAF) that mediate inflammatory responses in the disease. PAF is known to activate NF-κB, however, the mechanisms of PAF-induced inflammation are not fully defined. We have recently described a novel PAF-triggered pathway of NF-κB activation and IL-8 production in intestinal epithelial cells (IECs), requiring the pivotal role of the adaptor protein Bcl10 and its interactions with CARMA3 and MALT1. The current studies examined the potential role of the probiotic Lactobacillus acidophilus in reversing the PAF-induced, Bcl10-dependent NF-κB activation and IL-8 production in IECs. PAF treatment (5 µM×24 h) of NCM460 and Caco-2 cells significantly increased nuclear p65 NF-κB levels and IL-8 secretion (2-3-fold, P<0.05), compared to control, which were blocked by pretreatment of the cells for 6 h with L. acidophilus (LA) or its culture supernatant (CS), followed by continued treatments with PAF for 24 h. LA-CS also attenuated PAF-induced increase in Bcl10 mRNA and protein levels and Bcl10 promoter activity. LA-CS did not alter PAF-induced interaction of Bcl10 with CARMA3, but attenuated Bcl10 interaction with MALT1 and also PAF-induced ubiquitination of IKKγ. Efficacy of bacteria-free CS of LA in counteracting PAF-induced inflammatory cascade suggests that soluble factor(s) in the CS of LA mediate these effects. These results define a novel mechanism by which probiotics counteract PAF-induced inflammation in IECs.     

Crystal structure analysis of phosphatidylcholine-GM2-activator product complexes: evidence for hydrolase activity

GM2-activator protein (GM2AP) is a lysosomal lipid transfer protein with important biological roles in ganglioside catabolism, phospholipid metabolism, and T-cell activation. Previous studies of crystal structures of GM2AP complexed with the physiological ligand GM2 and platelet activating factor (PAF) have shown binding at two specific locations within the spacious apolar pocket and an ordering effect of endogenous resident lipids. To investigate the structural basis of phospholipid binding further, GM2AP was cocrystallized with phosphatidylcholine (PC), known to interact with GM2AP. Analysis of three crystal forms revealed binding of single chain lipids and fatty acids only and surprisingly not intact PC. The regions of best defined electron density are consistent with the presence of lyso-PC and oleic acid, which constitute deacylation products of PC. Their acyl tails are in stacking contact with shorter, less well-defined stretches of electron density that may represent resident fatty acids. The GM2AP associated hydrolytic activity that generates lyso-PC was further confirmed by mass spectrometry and enzymatic assays. In addition, we report the structures of (i) mutant Y137S, assessing the role of Tyr137 in lipid transfer via the hydrophobic cleft, and (ii) apo-mouse GM2AP, revealing a hydrophobic pocket with a constricted opening. Our structural results provide new insights into the biological functions of GM2AP. The combined effect of hydrolytic and lipid transfer properties has profound implications in cellular signaling.     

The GM2 activator protein, a novel inhibitor of platelet-activating factor.

The GM2 activator protein is required as a substrate-specific cofactor for beta-hexosaminidase A to hydrolyze GM2 ganglioside. The GM2 activator protein reversibly binds and solubilizes individual GM2 ganglioside molecules, making them available as substrate. Although GM2 ganglioside is the strongest binding ligand for the activator protein, it can also bind and transport between membranes a series of other glycolipids, even at neutral pH. Biosynthetic studies have shown that a large portion of newly synthesized GM2 activator molecules are not targeted to the lysosome, but are secreted and can then be recaptured by other cells through a carbohydrate independent mechanism. Thus, the GM2 activator protein may have other in vivo functions. We found that the GM2 activator protein can inhibit, through specific binding, the ability of platelet activating factor (PAF) to stimulate the release of intracellular Ca2+ pools by human neutrophils. PAF is a biologically potent phosphoacylglycerol. Inhibitors for PAF's role in the pathogenesis of inflammatory bowel disease and asthma have been sought as potential therapeutic agents. The inherent stability and protease resistance of the small, monomeric GM2 activator protein, coupled with the ability to produce large quantities of the functional protein in transformed bacteria, suggest it may serve as such an agent.     

Effects of Soluble Epoxide Hydrolase Deficiency on Acute Pancreatitis in Mice

Background

Acute pancreatitis (AP) is a frequent gastrointestinal disorder that causes significant morbidity, and its incidence has been progressively increasing. AP starts as a local inflammation in the pancreas that often leads to systemic inflammatory response and complications. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition in murine models has beneficial effects in inflammatory diseases, but its significance in AP remains unexplored.

Methodology/Principal Findings

To investigate whether sEH may have a causal role in AP we utilized Ephx2 knockout (KO) mice to determine the effects of sEH deficiency on cerulein- and arginine-induced AP. sEH expression increased at the protein and messenger RNA levels, as well as enzymatic activity in the early phase of cerulein- and arginine-induced AP in mice. In addition, amylase and lipase levels were lower in cerulein-treated Ephx2 KO mice compared with controls. Moreover, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B and IL-6 were lower in cerulein-treated Ephx2 KO mice compared with controls. Further, Ephx2 KO mice exhibited decreased cerulein- and arginine-induced NF-κB inflammatory response, MAPKs activation and decreased cell death. Conclusions -These findings demonstrate a novel role for sEH in the progression of cerulein- and arginine-induced AP.     

Serum PAF acetylhydrolase increases during neonatal maturation

Acetylhydrolase is an acid-labile, 43 kd protein that catalyzes the degradation of platelet activating factor (PAF), a potent phospholipid inflammatory mediator, to its biologically inactive metabolite lysoPAF. PAF has a short half-life, thus acetylhydrolase plays an important role in its regulation. Since previous work suggests that PAF may be involved in certain neonatal diseases such as necrotizing enterocolitis, we studied the effect of age on acetylhydrolase activity. Serum acetylhydrolase activity was quantified using radio-labelled PAF and measuring reaction products. Serum samples were obtained prospectively from 70 subjects ranging in age from 4 hr to 48 yr. Acetylhydrolase activity was lower for newborns (< 3 wk) than all other age ranges (8.2 ± 1.4 nmole/ml/min vs 30.0 ± 1.6 nmole/ml/min, p < .01). Furthermore, enzyme activity increased linearly with respect to the natural logarithm of age from 0 days to 6 weeks (r = 0.65. p < .001). By 6 weeks of life acetylhydrolase activity approached values of older children and adults. Newborn acetylhydrolase activity was similar between term and preterm infants (8.6 ± 1.9 nmole/ml/min vs 7.2 ± 2.4 nmole/ml/min, p = NS). We conclude that acetylhydrolase activity is low in human neonates and increases during the first 6 weeks of life. These results suggest that newborn infants may be at increased risk for pathophysiologic processes mediated by PAF.     

Worms,flies and four-legged friends: the applicability of biological models to the understanding of intestinal inflammatory diseases

Diseases of intestinal inflammation, including Crohn’s disease, ulcerative colitis and necrotizing enterocolitis, cause substantial acute and chronic disability in a large proportion of the population. Crohn’s disease and ulcerative colitis, which are collectively referred to as inflammatory bowel disease (IBD), lead to recurrent episodes of intestinal dysfunction and systemic illness, whereas necrotizing enterocolitis is characterized by the development of dramatic and all too often fatal intestinal necrosis in infants. To determine the molecular underpinnings of these disorders, investigators have explored a variety of animal models that vary widely in their complexity. These experimental systems include the invertebrate nematode Caenorhabditis elegans, the more complex invertebrate Drosophila melanogaster, and vertebrate systems including mice, rats and other mammals. This review explores the experimental models that are used to mimic and evaluate the pathogenic mechanisms leading to these diseases of intestinal inflammation. We then highlight, as an example, how the use of different experimental models that focus on the role of Toll-like receptor 4 (TLR4) signaling in the gut has revealed important distinctions between the pathogenesis of IBD and necrotizing enterocolitis. Specifically, TLR4-mediated signaling plays a protective role in the development of Crohn’s disease and ulcerative colitis, whereas this signaling pathway plays a causative role in the development of necrotizing enterocolitis in the newborn small intestine by adversely affecting intestinal injury and repair mechanisms.     

Serum PAF acetylhydrolase increases during neonatal maturation

Acetylhydrolase is an acid-labile, 43 kd protein that catalyzes the degradation of platelet activating factor (PAF), a potent phospholipid inflammatory mediator, to its biologically inactive metabolite lysoPAF. PAF has a short half-life, thus acetylhydrolase plays an important role in its regulation. Since previous work suggests that PAF may be involved in certain neonatal diseases such as necrotizing enterocolitis, we studied the effect of age on acetylhydrolase activity. Serum acetylhydrolase activity was quantified using radio-labelled PAF and measuring reaction products. Serum samples were obtained prospectively from 70 subjects ranging in age from 4 hr to 48 yr. Acetylhydrolase activity was lower for newborns (less than 3 wk) than all other age ranges (8.2 +/- 1.4 nmole/ml/min vs 30.0 +/- 1.6 nmole/ml/min, p less than .01). Furthermore, enzyme activity increased linearly with respect to the natural logarithm of age from 0 days to 6 weeks (r = 0.65, p less than .001). By 6 weeks of life acetylhydrolase activity approached values of older children and adults. Newborn acetylhydrolase activity was similar between term and preterm infants (8.6 +/- 1.9 nmole/ml/min vs 7.2 +/- 2.4 nmole/ml/min, p = NS). We conclude that acetylhydrolase activity is low in human neonates and increases during the first 6 weeks of life. These results suggest that newborn infants may be at increased risk for pathophysiologic processes mediated by PAF.     

Lipoprotein-associated phospholipase A(2) interacts with phospholipid vesicles via a surface-disposed hydrophobic α-helix

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) plays important roles in both the inhibition and promotion of inflammation in human disease. It catalyzes the hydrolytic inactivation of plasma platelet activating factor (PAF) and is also known as PAF acetylhydrolase. High levels of PAF are implicated in a variety of inflammatory diseases such as asthma, necrotizing enterocolitis, and sepsis. Lp-PLA(2) also associates with lipoproteins in human plasma where it hydrolyzes oxidized phospholipids to produce pro-inflammatory lipid mediators that can promote inflammation and the development of atherosclerosis. Lp-PLA(2) plasma levels have recently been identified as a biomarker of vascular inflammation, atherosclerotic vulnerability, and future cardiovascular events. The enzyme is thus a prominent target for the development of inflammation and atherosclerosis-modulating therapeutics. While the crystallographically determined structure of the enzyme is known, the enzyme's mechanism of interaction with PAF and the function-modulating lipids in lipoproteins is unknown. We have employed peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to characterize the association of Lp-PLA(2) with dimyristoylphosphatidylcholine (DMPC) vesicles and found that specific residues 113-120 in one of the enzyme's surface-disposed hydrophobic α-helices likely mediate liposome binding.     

Evidence for lipid packaging in the crystal structure of the GM2-activator complex with platelet activating factor

GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2A crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement of two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals' contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.     

Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C₁₇H₂₇NO_9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.     

In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits,Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.     

Erythropoietin Protects Epithelial Cells from Excessive Autophagy and Apoptosis in Experimental Neonatal Necrotizing Enterocolitis

Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of preterm infants. Increased intestinal epithelium permeability is an early event in NEC pathogenesis. Autophagy and apoptosis are induced by multiple stress pathways which may impact the intestinal barrier, and they have been associated with pathogenesis of diverse gastrointestinal diseases including inflammatory bowel disease. Using both in vitro and in vivo models, this study investigates autophagy and apoptosis under experimental NEC stresses. Furthermore this study evaluates the effect of erythropoietin (Epo), a component of breast milk previously shown to decrease the incidence of NEC and to preserve intestinal barrier function, on intestinal autophagy and apoptosis. It was found that autophagy and apoptosis are both rapidly up regulated in NEC in vivo as indicated by increased expression of the autophagy markers Beclin 1 and LC3II, and by evidence of apoptosis by TUNEL and cleaved caspase-3 staining. In the rat NEC experimental model, autophagy preceded the onset of apoptosis in intestine. In vitro studies suggested that Epo supplementation significantly decreased both autophagy and apoptosis via the Akt/mTOR signaling pathway and the MAPK/ERK pathway respectively. These results suggest that Epo protects intestinal epithelium from excessive autophagy and apoptosis in experimental NEC.     

Study of Interaction of GM2 Activator Protein with GM2 Using Circular Dichroism and Fluorescence Spectroscopy

GM2 activator protein (GM2AP) is a cofactor for stimulating the enzymatic hydrolysis of the glycolipid GM2 by -hexosaminidase A to produce GM3. We have examined the conformation of GM2AP before and after its interaction with GM2, GM3, and GA2 using circular dichroism and fluorescence spectroscopy techniques. In the presence of GM2, a blue shift of the fluorescence emission maximum and a strong decrease of molar ellipticity values in circular dichroism spectra were observed only at pH 4.5 and at GM2/GM2AP molar ratio higher than 10:1 (up to 50:1). These results suggest that GM2AP assumed a more organized -helical conformation with the tryptophan residues moving from the polar medium toward the hydrophobic environment of the protein. The conformation of GM2AP in the presence of the downstream reaction product, GM3, or a less favorable substrate, GA2, clearly differed from that in the presence of GM2. The relationships between spectroscopic changes and enzymatic activity, herein discussed, strongly suggest that the specific conformation exhibited by GM2AP in the presence of GM2 is functional to serve as an activator for the enzymatic hydrolysis of GM2.     

Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity

Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids.     

Ligand Extraction Properties of the GM2 Activator Protein and Its Interactions with Lipid Vesicles

The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.     

Effect of GM2 activator protein on the enzymatic hydrolysis of phospholipids and sphingomyelin

GM2 activator protein (GM2AP) is a specific protein cofactor that stimulates the enzymatic hydrolysis of the GalNAc from GM2, a sialic acid containing glycosphingolipid, both in vitro and in lysosomes. While phospholipids together with glycosphingolipids are important membrane constituents, little is known about the possible effect of GM2AP on the hydrolysis of phospholipids. Several recent reports suggest that GM2AP might have functions other than stimulating the conversion of GM2 into GM3 by beta-hexosaminidase A, such as inhibiting the activity of platelet activating factor and enhancing the degradation of phosphatidylcholine by phospholipase D (PLD). We therefore examined the effect of GM2AP on the in vitro hydrolyses of a number of phospholipids and sphingomyelin by microbial (Streptomyces chromofuscus) and plant (cabbage) PLD. GM2AP, at the concentration as low as 1.08 microM (1 microg/50 microl) was found to inhibit about 70% of the hydrolyses of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol by PLD, whereas the same concentration of GM2AP only inhibited about 20-25% of the hydrolysis of sphingomyelin by sphingomyelinase and had no effect on the hydrolysis of sphingosylphosphorylcholine by PLD. Thus, GM2AP exerts strong and broad inhibitory effects on the hydrolysis of phospholipids carried out by plant and microbial PLDs. High ammonium sulfate concentration (1.6 M or 21.1%) masks this inhibitory effect, possibly due to the alteration of the ionic property of GM2AP.     

Ganglioside GM3 suppresses lipopolysaccharide‐induced inflammatory responses in rAW 264.7 macrophage cells through NF‐κB,AP‐1, and MAPKs signaling

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase‐2 (COX‐2) protein and mRNA levels in lipopolysaccharide (LPS)‐activated RAW 264.7 cells in a dose‐dependent manner. Moreover, GM3 inhibited the expression and release of pro‐inflammatory cytokines of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS‐induced nuclear translocation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and activator protein (AP)‐1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen‐activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS‐activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS‐induced inflammatory response in RAW 264.7 macrophages by suppression of NF‐κB, AP‐1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.     

The trafficking of prosaposin (SGP-1) and GM2AP to the lysosomes of TM4 Sertoli cells is mediated by sortilin and monomeric adaptor proteins

Prosaposin (SGP-1) and GM2 activator protein (GM2AP) are soluble sphingolipid activator proteins (SAPs) that are targeted to the lysosomal compartment of Sertoli cells to aid hydrolases in the breakdown of glycosphingolipids. To reach the lysosome, most soluble proteins must interact with the mannose 6-phosphate receptor (MPR). To be sorted from the Golgi, the MPR must bind to the Golgi associated, gamma-adaptin homologous, ARF binding proteins (GGAs), a group of monomeric adaptor proteins responsible for the recruitment of clathrin. It is well established, however, that the lysosomes of I-cell disease (ICD) patients have near normal levels of several lysosomal proteins, including prosaposin and GM2AP. ICD results from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases. Thus, prosaposin and GM2AP can traffic to lysosomes in a MPR independent manner. Previous work has demonstrated that an interaction with sphingomyelin in the Golgi membrane is necessary for the targeting of prosaposin by an unknown receptor. Using a TM4 Sertoli cell line, we tested the hypothesis that prosaposin and GM2AP are targeted to the lysosomal compartment via the sortilin receptor, which has been recently shown to have a GGA binding motif. Interestingly, dominant-negative GGAs, unable to bind clathrin to shuttle from the Golgi, prevented the trafficking of prosaposin and GM2AP to lysosomes. A dominant negative construct of sortilin lacking the GGA binding domain retained prosaposin and GM2AP in the Golgi. In conclusion, our results showed that the trafficking of prosaposin and GM2AP to the lysosome is dependent on sortilin.     

The enzyme-binding region of human GM2-activator protein

The GM2-activator protein (GM2AP) is an essential cofactor for the lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (HexA). It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interface. Functional deficiencies in this protein result in a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. In order to elucidate this cofactor's mode of action and identify the surface region of GM2AP responsible for binding to HexA, we designed several variant forms of this protein and evaluated the consequences of these mutations for lipid- and enzyme-binding properties using a variety of biophysical and functional studies. The point mutants D113K, M117V and E123K showed a drastically decreased capacity to stimulate HexA-catalysed GM2 degradation. However, surface plasmon resonance (SPR) spectroscopy showed that the binding of these variants to immobilized lipid bilayers and their ability to solubilize lipids from anionic vesicles were the same as for the wild-type protein. In addition, a fluorescence resonance energy transfer (FRET)-based assay system showed that these variants had the same capacity as wild-type GM2AP for intervesicular lipid transfer from donor to acceptor liposomes. The concentration-dependent effect of these variants on hydrolysis of the synthetic substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-6-sulfo-beta-D-glucopyranoside (MUGS) indicated a weakened association with the enzyme's alpha subunit. This identifies the protein region affected by these mutations, the single short alpha helix of GM2AP, as the major determinant for the interaction with the enzyme. These results further confirm that the function of GM2AP is not restricted to a biological detergent that simply disrupts the membrane structure or lifts the substrate out of the lipid plane. In contrast, our data argue in favour of the critical importance of distinct activator-hexosaminidase interactions for GM2 degradation, and corroborate the view that the activator/lipid complex represents the true substrate for the degrading enzyme.