A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol

Okamura, Nobuyuki; Kiuchi, Sachiko; Tamba, Michiko; Kashima, Takayuki; Hiramoto, Shinsuke; Baba, Tadashi; Dacheux, Françoise; Dacheux, Jean‐Louis; Sugita, Yoshiki; Jin, Yin‐Zhe

doi:10.1016/s1388-1981(99)00070-0

Cited by 179 publications

(135 citation statements)

References 45 publications

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“…Proteins in several classes are known to interact with sperm in mammals; these classes are also found among Drosophila Acps: lipases, lectins, and CRISPs. In mammals, secreted lipid carriers and modifiers in the seminal plasma HE1, clusterin, lipocalin-type prostaglandin D synthase, and cholesterol-binding protein, comprising up to 30% of the total seminal fluid secretions, function in lipid binding͞modification in mammalian seminal fluid (61,62). It is possible that the six Acp lipases may also play a role in lipid modification or in providing energy to the sperm, which may affect sperm motility and͞or viability.…”

Section: Discussionmentioning

confidence: 99%

Comparative structural modeling and inference of conserved protein classes in Drosophila seminal fluid

Mueller

Ripoll

Aquadro

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

121

157

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The constituents of seminal fluid are a complex mixture of proteins and other molecules, most of whose functions have yet to be determined and many of which are rapidly evolving. As a step in elucidating the roles of these proteins and exposing potential functional similarities hidden by their rapid evolution, we performed comparative structural modeling on 28 of 52 predicted seminal proteins produced in the Drosophila melanogaster male accessory gland. Each model was characterized by defining residues likely to be important for structure and function. Comparisons of known protein structures with predicted accessory gland proteins (Acps) revealed similarities undetectable by primary sequence alignments. The structures predict that Acps fall into several categories: regulators of proteolysis, lipid modifiers, immunity͞ protection, sperm-binding proteins, and peptide hormones. The comparative structural modeling approach indicates that major functional classes of mammalian and Drosophila seminal fluid proteins are conserved, despite differences in reproductive strategies. This is particularly striking in the face of the rapid protein sequence evolution that characterizes many reproductive proteins, including Drosophila and mammalian seminal proteins.

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Section: Discussionmentioning

confidence: 99%

Comparative structural modeling and inference of conserved protein classes in Drosophila seminal fluid

Mueller

Ripoll

Aquadro

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

121

157

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“…To this end, we purified wild-type and mutant NPC2 proteins, starting from a culture medium into which cells had released NPC2. Previous studies of NPC2 used NPC2-conditioned media (7) or NPC2 purified from porcine epididymal fluid (10). These procedures are not practical for studying a substantial number of mutant proteins, so we developed a rapid affinity-purification procedure.…”

Section: Purification Of Functional Tagged Npc2 Protein and Stoichiommentioning

confidence: 99%

“…The porcine HE1 homolog was reported to bind cholesterol with micromolar affinity (K d ϭ 2.3 M; ref. 10), information that was crucial in identifying HE1 as a candidate npc2 gene product (7). However, the similarity between the reported K d and the solubility of cholesterol in aqueous solution (5 M, Merck Index) warrants reexamination of the strength and specificity of this interaction.…”

mentioning

confidence: 99%

The integrity of a cholesterol-binding pocket in Niemann–Pick C2 protein is necessary to control lysosome cholesterol levels

Binkley

Sidow

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

182

196

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The neurodegenerative disease Niemann-Pick Type C2 (NPC2) results from mutations in the NPC2 (HE1) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (K d ‫؍‬ 30 -50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.point mutants ͉ secretion ͉ protein evolution N iemann-Pick Type C (NPC) is an autosomal-recessive disorder characterized by progressive ataxia, dystonia, and dementia (1). Substantial cell death, particularly in the cerebellum, accounts for some of the symptoms. At the cellular level, mutant cells accumulate cholesterol and other lipids in aberrant compartments with features of late endosomes and lysosomes, and the normal homeostatic response to this excess cholesterol is abolished (2-4).Our understanding of the molecular basis of this disease has progressed substantially over the last several years, because the two genes damaged by NPC mutations, NPC1 and NPC2, have been identified (5-7). NPC1 encodes a multiple-membranespanning protein containing sequences similar to the sterol cleavage activating protein (SCAP) regulator of cholesterol metabolism and the receptor Patched, a transducer of the Hedgehog family of protein signals. Mutations in NPC1 are responsible for Ϸ95% of the NPC cases (8). The exact role that NPC1 plays in maintaining normal levels of cholesterol in late endosomes and lysosomes remains mysterious. Even less is known about the NPC2 [human epididymis (HE1)] protein, the loss of which is responsible for 5% of NPC cases.NPC2 is a small secreted glycoprotein originally identified as a transcript enriched in the HE1 (9). The porcine HE1 homolog was reported to bind cholesterol with micromolar affinity (K d ϭ 2.3 M; ref. 10), information that was crucial in identifying HE1 as a candidate npc2 gene product (7). However, the similarity between the reported K d and the solubility of cholesterol in aqueous solution (5 M, Merck Index) warrants reexamination of the strength and specificity of this interaction. If the binding is meaningful, NPC2 activity could be altered when cholesterol is bound, thus serving as a sensor of cholesterol levels, accessibility, or location. Alternatively, NPC2 could process or transport cholesterol. Resolving these questions is crucial for determining why cholesterol accumulates in people lacking functional NPC2 protein and for designing rational treatment strategies.In this report, we combine in vitro binding experiments, genetic analysis, a quantitative cell-based ...

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“…NPC2 was originally identified as the major secretory protein of the epididymis (27). It is known to bind cholesterol and can be secreted from cells as cholesterol bound (28,29). It was, therefore, of interest to analyze the secretion of NPC2 from normal and NPC1 Ϫ/Ϫ astrocytes.…”

Section: Fig 5 Secretion Of De Novo Sterols Is Not Dependent On Npcmentioning

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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A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol

Cited by 179 publications

References 45 publications

Comparative structural modeling and inference of conserved protein classes in Drosophila seminal fluid

Comparative structural modeling and inference of conserved protein classes in Drosophila seminal fluid

The integrity of a cholesterol-binding pocket in Niemann–Pick C2 protein is necessary to control lysosome cholesterol levels

Secretion of Sterols and the NPC2 Protein from Primary Astrocytes

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