Structure of a cholesterol-binding protein deficient in Niemann–Pick type C2 disease

Friedland, Natalia; Liou, Heng-Ling; Lobel, Peter; Stock, Ann

doi:10.1073/pnas.0437840100

Cited by 291 publications

(275 citation statements)

References 31 publications

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“…Der p2, p1, f 2), GM2-activator protein, Npc2, and their orthologs (16). The recent publication of the crystal structure of recombinant MD-2 (17) confirms earlier structural models of MD-2 that were based on the solved structures of other ML domain proteins (31)(32)(33)(34)(35)(36)(37). A hallmark of MD-2, as well as other ML domain proteins, is the presence of a deep, hydrophobic cavity that can expand to accommodate specific (glyco)lipid ligands.…”

Section: Discussionsupporting

confidence: 59%

Isolation of Monomeric and Dimeric Secreted MD-2

Teghanemt

Widstrom

Gioannini

et al. 2008

Journal of Biological Chemistry

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Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E⅐MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E⅐sCD14 to form monomeric E⅐MD-2 and activate HEK293/ TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E⅐MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E⅐CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.Invasion by Gram-negative bacteria (GNB) 3 is specifically detected and responded to by mammals through mobilization of the innate immune system. In many strains and species of GNB, this process depends on host recognition of and response to the unique complex glycolipid, endotoxin (lipooligosaccharide (LOS) or lipopolysaccharide (LPS)), that comprises much of the outer leaflet of the outer membrane of GNB (1, 2). Minute amounts of endotoxin (E), presented either as an integral part of the outer membrane of GNB or as large aggregates of extracted and purified endotoxin, can stimulate pro-inflammatory responses (2-5). This sensitivity depends upon an ordered series of endotoxin-protein and protein-protein interactions that include the host proteins LPS-binding protein (LBP), membrane-bound or soluble (s)CD14, secreted or TLR4-associated MD-2, and TLR4 (6 -8). Engagement of endotoxin-rich membranes or isolated endotoxin aggregates by LBP facilitates the extraction of endotoxin monomers by CD14 to form monomeric E⅐CD14 complexes that are the most efficient substrate for transfer of endotoxin to MD-2 (4, 6, 9). The monomeric E⅐MD-2 complex is necessary and sufficient to induce TLR4-dependent cell activation by endotoxin (4, 6, 10). MD-2 associates noncovalently with the N-terminal ectodomain of TLR4 and plays a pivotal role not only in TLR4 activation but also in trafficking of TLR4 to the cell surface (7, 11-13). MD-2 also likely interacts transiently with CD14 facilitating transfer of endotoxin monomer from CD14 to MD-2 and, at high molar excess of CD14, reverse transfer of endotoxin from MD-2 to CD14 (14). Simultaneous engagement by MD-2...

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Section: Discussionsupporting

confidence: 59%

Isolation of Monomeric and Dimeric Secreted MD-2

Teghanemt

Widstrom

Gioannini

et al. 2008

Journal of Biological Chemistry

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“…However, a corresponding increase in the [ 3 H]cholesterol radioactivity in the relevant fractions was not observed. The amount of NPC2 protein may have been too small despite the overexpression to introduce a [ 3 H]cholesterol signal to exceed the background radioactivity as the stoichiometry of NPC2 and sterol is apparently 1:1 (36). Alternatively, NPC2 may not be cholesterol-associated when secreted from astrocytes or the [ 3 H]cholesterol radiolabel might have been too poorly represented in the pool from which NPC2 took its cholesterol.…”

Section: Discussionmentioning

confidence: 99%

Secretion of Sterols and the NPC2 Protein from Primary Astrocytes

Mutka

Lusa

Linder

et al. 2004

Journal of Biological Chemistry

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Astrocytes secrete cholesterol in lipoprotein particles.Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1 ؊/؊ ) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1 ؊/؊ astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors. The brain is the most cholesterol-rich organ in the body, containing roughly 25% of the unesterified cholesterol present in the whole individual. The input of cholesterol into the brain comes entirely, or almost entirely, from in situ synthesis because blood lipoproteins do not cross the blood-brain-barrier (1). Glial cells are thought to be responsible for a large part of this biosynthetic activity. Most of the sterol in the brain is acquired during myelination in the early stages of development and is produced by oligodendrocytes (2).Astrocytes, the most abundant glial cells, are intimately associated with neuronal synapses and secrete cholesterol in lipoprotein particles. Astrocytes have been proposed to provide cholesterol for synapse formation (3) as well as to participate in the recycling of cholesterol after injury (4). Nascent lipoproteins isolated from neonatal mouse astrocytes are a heterogeneous population of particles in the size range of plasma high density lipoproteins and appear to be composed of two separate classes that contain either apolipoprotein E or J (5).Niemann-Pick type C disease (NPC) 1 is an inherited, fatal neurodegenerative disorder in which large amounts of cholesterol and sphingolipids accumulate intracellularly within late endocytic organelles. The disease is caused by mutations in either the NPC1 or NPC2 gene (6). The NPC1 protein is a late endosomal membrane protein harboring a sterol-sensing domain, whereas NPC2 is a secretory protein that has cholesterol binding properties and uses the mannose 6-phosphate marker for late endosomal targeting (7). The exact functions of the proteins remain unknown, but genetic evidence suggests that they function in concert to facilitate lysosomal lipid egress (8).In the NPC1 Ϫ/Ϫ mouse astrocytes are considered to contribute to neurodegeneration (9, 10). Interestingly, NPC1 Ϫ/Ϫ astrocytes were shown t...

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“…Glycosylation has been shown to be essential for human NPC2 function [48] and a conserved N-glycosylation site was predicted in the S. mansoni NPC2 (Figure 3). Furthermore, molecular modeling of S. mansoni NPC2 generated a predicted structure similar to human NPC2 [49] that included conservation of the order of β-sheets, Cys-Cys bonds, and localization of the residues implicated in cholesterol binding to a hydrophobic pocket in the protein (Figure 3) suggesting a similar cholesterol-binding function for the S. mansoni NPC2 protein. One possible explanation for the presence of NPC2 in ESP is to facilitate the movement of cholesterol (which eggs must scavenge).…”

Section: Functions Of Proteins Found In Espmentioning

confidence: 99%

Proteomic analysis of Schistosoma mansoni egg secretions

Cass

Johnson

Califf

et al. 2007

Molecular and Biochemical Parasitology

194

196

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Schistosomiasis remains a largely neglected, global health problem. The morbid pathology of the disease stems from the host's inflammatory response to parasite eggs trapped in host tissues. Long term host/parasite survival is dependent upon the successful modulation of the acute pathological response, which is induced by egg antigens. In this study, using Multidimensional Protein Identification Technology, we identified the Schistosoma mansoni egg secretome consisting of 188 proteins. Notably we identified proteins involved in redox balance, molecular chaperoning and protein folding, development and signaling, scavenging and metabolic pathways, immune response modulation, and 32 novel, previously uncharacterized schistosome proteins. We localized a subset of previously-characterized schistosome proteins identified in egg secretions in this study, to the surface of live S. mansoni eggs using the circumoval precipitin reaction. The identification of proteins actively secreted by live schistosome eggs provides important new information for understanding immune modulation and the pathology of schistosomiasis.

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Structure of a cholesterol-binding protein deficient in Niemann–Pick type C2 disease

Cited by 291 publications

References 31 publications

Isolation of Monomeric and Dimeric Secreted MD-2

Isolation of Monomeric and Dimeric Secreted MD-2

Secretion of Sterols and the NPC2 Protein from Primary Astrocytes

Proteomic analysis of Schistosoma mansoni egg secretions

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