Apolipoprotein D--an atypical apolipoprotein.

The structure of ApoD and its sites of synthesis have been discovered. These characteristics differ from those of the other apolipoproteins. The role of ApoD in the plasma lipoprotein system remains to be discovered, but the recent, rapid increase in our knowledge of this protein suggests that it plays an important role in the homeostasis or housekeeping of probably all organs. One of its functions is likely to be the transport of a hydrophobic ligand (a lipid) in a one-to-one molar ratio with itself. This transport is likely to occur unidirectionally between neighboring cells in an organ, and between perivascular cells and the blood circulation. The chemical structure of the natural ligand, or ligands, of ApoD in normal cells in vivo or in culture is not known, but ApoD has been shown to bind some steroids and bilirubin. Remarkable upregulation of synthesis of ApoD has been observed during regeneration of injured peripheral nerves. Perhaps the physiologic role of ApoD will prove to be more interesting and of equal importance in biology to the roles of the other apolipoproteins in cardiovascular disease.     

From pharmacotherapy to pathophysiology: emerging mechanisms of apolipoprotein D in psychiatric disorders

Apolipoprotein D (apoD) is an atypical plasma apolipoprotein and, based on its primary structure, it is a member of the lipocalin protein superfamily. Lipocalins have been extensively used as disease markers and, accordingly, apoD has become increasingly recognized as an important factor in the pathology of human neurodegenerative and neuropsychiatric disorders. ApoD expression is increased in the plasma and brains of subjects with schizophrenia and bipolar disorder, suggesting that it acts as a marker for disease pathology. ApoD also exhibits complex regulation by antipsychotic drug treatment and may represent a distinguishing mechanism of typical versus atypical drugs. The precise role of apoD in the CNS and disease remains to be elucidated, but recent findings have suggested that it plays an important role in the regulation of arachidonic acid signaling and metabolism providing further support for phospholipid membrane pathology in schizophrenia.     

Studies on the isolation and partial characterization of apolipoprotein D and lipoprotein D of human plasma.

This report describes further studies on the characterization of apolipoprotein D (ApoD), a recently recognized human plasma apolipoprotein, and presents results on the isolation and distribution of its lipoprotein form, lipoprotein D (LP-D). ApoD, isolated by a procedure combining hydroxylapatite and Sephadex G-100 column chromatography, migrated on 7% polyacrylamide gel as a single band with a mobility intermediate between those of A-II and C-II polypeptides. On double diffusion and immunoelectrophoresis, ApoD reacted only with antiserum to ApoD. It was characterized by the presence of all common amino acids including half-cystine. The amino terminal acid was blocked. Carbohydrate analysis demonstrated that ApoD is a glycoprotein with glucose, mannose, galactose, glucosamine, and sialic acid accounting for 18% of the dry weight of ApoD. The estimated molecular weight of ApoD IS 22 100. ApoD occurs in the serum as a lipoprotein which was isolated from high density lipoproteins3 by two different chromatographic procedures. In the first procedure, high density lipoproteins3 were treated with neuraminidase and chromatographed on concanavlin A. The retained fraction containing LP-D was purified by hydroxylapatite column chromatography. Alternatively, LP-D was isolated by a procedure combining chromatography of high density lipoproteins3 or whole serum on an immunosorber containing antibodies to ApoD, and hydroxylapatite column chromatography. LP-D displayed a single, symmetrical boundary in the analytical ultracentrifuge and a single band on 7% polyacrylamide gel electrophoresis. When injected into rabbits it produced antisera that reacted only with ApoD. On immunoelectrophoresis LP-D had a mobility different from that of lipoprotein A (LP-A). A direct immunological comparison of LP-D and LP-A showed a reaction of nonidentity. LP-D consists of 65-75% protein and 25-35% lipid. The lipid moiety contains cholesterol, cholesterol ester, triglyceride, and phospholipid. The phospholipid. composition is characterized by a relative high content of lysolecithin and sphingomyelin and a relatively low content of lecithin. We have concluded from these studies that ApoD is a unique apolipoprotein that exists in the form of a distinct lipoprotein family with a macromolecular distribution extending from very low density lipoproteins into very high density lipoproteins, but with a maximum concentration in high density lipoproteins3 and a minimum concentration in high density lipoproteins.     

AP-1在腋臭患者发病过程中的作用

目的：腋臭是美容整形外科的常见病,目前发病机制尚不明确,已证实人体大汗腺中的载脂蛋白D（ApoD）在腋臭患者大汗腺中高表达,并且与腋臭的发生密切相关。探明ApoD在大汗腺细胞中的信号转导通路,可以进一步明确其在腋臭发病过程中的作用机制。JNK信号转导通路与多种疾病的发生有关。课题组前期已经做了JNKl对ApoD调控作用的相关研究,证明了在腋臭发病过程中JNK1是通过调控ApoD的转录来上调ApoD的表达。本实验在课题组前期研究基础上,探讨JNKl下游转录因子AP-1是否在JNKl上调ApoD通路中发挥作用。方法：取腋臭志愿者腋区皮肤组织,进行汗腺细胞培养。把汗腺细胞分为5．二氢睾酮处理组、5-二氢睾酮联合姜黄素处理组和空白对照3个组,用姜黄素抑制AP-1的活性,通过Real．timePCR实验方法检测ApoD在姜黄素抑制下的表达变化。结果：在姜黄素的抑制下,ApoD表达明显降低。在体外培养汗腺细胞加入5．二氢睾酮联合姜黄素处理后,ApoD的表达量在mRNA水平低于单独的5-二氢睾酮处理组和正常对照组（P〈0．05）。结论：姜黄素抑制了AP．1的活化导致ApoD的表达降低。在腋臭的发病过程中,JNKl的下游转录因子AP-1对ApoD有明显的上调作用。AP-1可能在JNKl上调ApoD这条通路中扮演了很重要的角色,它可能是JNK1和ApoD的中间转录因子。     

Expression of EphA receptors and ligands during chick cerebellar development

Apolipoprotein D (ApoD) is a secreted protein that belongs to the lipocalin family. We describe the expression pattern of ApoD during mouse embryogenesis by in situ hybridization using RNA probes. ApoD is expressed at E9 in mesenchymal cells in the rombencephalic–mesencephalic region. At E9.5 the cephalic ApoD-positive cells appear in the mesenchyme, and at later stages (starting at E10.5) ApoD expression is seen in meninges. Within the neuroepithelium, ApoD is expressed in pericytes surrounding brain and spinal cord capillaries from E10.5 to birth. Other places of expression of ApoD are the mesenchyme surrounding the olfactory epithelium and semicircular canals, as well as chondroblasts of skull and vertebrae.     

Antioxidant activities of recombinant amphioxus (<Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>) apolipoprotein D

Apolipoprotein D (ApoD), a member of lipocalin, has been recently shown to be involved in regulating protection from oxidative stress. The absence of ApoD in mouse and Drosophila can reduce the resistance to oxidative stress and shorten lifespan. However, little information is available regarding the expression in vitro of ApoD and its biochemical properties. Amphioxus (Branchiostoma belcheri) ApoD, BbApoD, is an archetype of vertebrate ApoD proteins. In this study, the prokaryotic expression plasmid pET32a–BbApoD was constructed and recombinant BbApoD expressed in Escherichia coli BL21 and purified. Antioxidation assays showed that the recombinant BbApoD protein had the capacities to scavenge hydroxyl radicals (≥240 μg/ml) and to prevent nicking of the supercoiled DNA (≥100 μg/ml) in vitro, providing a biochemical evidence for antioxidant role of ApoD. This supports the notion that ApoD is part of the mechanisms regulating protection from oxidative stresses.     

Solubility engineering and crystallization of human apolipoprotein D

Human apolipoprotein D (ApoD) is a physiologically important member of the lipocalin protein family that was discovered as a peripheral subunit of the high-density lipoprotein (HDL) but is also abundant in other body fluids and organs, including neuronal tissue. Although it has been possible to produce functional ApoD in the periplasm of Escherichia coli and to demonstrate its ligand-binding activity for progesterone and arachidonic acid, the recombinant protein suffers from a pronounced tendency to aggregate and to adsorb to vessel surfaces as well as chromatography matrices, thus hampering further structural investigation. Here, we describe a systematic mutagenesis study directed at presumably exposed hydrophobic side chains of the unglycosylated recombinant protein. As a result, one ApoD mutant with just three new amino acid substitutions--W99H, I118S, and L120S--was identified, which exhibits the following features: (1) improved yield upon periplasmic biosynthesis in E. coli, (2) elution as a monomeric protein from a gel permeation chromatography column, and (3) unchanged binding activity for its physiological ligands. In addition, the engineered ApoD was successfully crystallized (space group I4 with unit cell parameters a = 75.1 A, b = 75.1 A, c = 166.0 A, alpha = beta = gamma = 90 degrees), thus demonstrating its conformationally homogeneous behavior and providing a basis for the future X-ray structural analysis of this functionally still puzzling protein.     

Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan

Increased Apolipoprotein D (ApoD) expression has been reported in various neurological disorders, including Alzheimer's disease, schizophrenia, and stroke, and in the aging brain . However, whether ApoD is toxic or a defense is unknown. In a screen to identify genes that protect Drosophila against acute oxidative stress, we isolated a fly homolog of ApoD, Glial Lazarillo (GLaz). In independent transgenic lines, overexpression of GLaz resulted in increased resistance to hyperoxia (100% O(2)) as well as a 29% extension of lifespan under normoxia. These flies also displayed marked improvements in climbing and walking ability after sublethal exposure to hyperoxia. Overexpression of Glaz also increased resistance to starvation without altering lipid or protein content. To determine whether GLaz might be important in protection against reperfusion injury, we subjected the flies to hypoxia, followed by recovery under normoxia. Overexpression of GLaz was protective against behavioral deficits caused in normal flies by this ischemia/reperfusion paradigm. This and the accompanying paper by Sanchez et al. (in this issue of Current Biology) are the first to manipulate the levels of an ApoD homolog in a model organism. Our data suggest that human ApoD may play a protective role and thus may constitute a therapeutic target to counteract certain neurological diseases.     

Increased JNK1 activity contributes to the upregulation of ApoD in the apocrine secretory gland cells from axillary osmidrosis

Axillary osmidrosis is a benign disorder that causes functional and emotional problems in Asian patients. Recently, ApoD has been identified as an axillary odorant binding protein. The present study was designed to compare the expression of ApoD in normal and osmidrosis subjects. Compared with the normal subjects, osmidrosis subjects had a higher expression of AR and ApoD in the apocrine samples, both at mRNA and protein level. Further study showed that, consistent with the increased ApoD and AR, phosphorylated JNK1 was higher in apocrine samples from axillary osmidrosis subjects, while with no obvious differences of the total expression of JNK1. In the cultured apocrine epithelial cells from normal subjects, 5α-dihydrotestosterone (5α-DHT) increased the expression of ApoD in a dose dependent manner, which can be inhibited by the JNK1 inhibitor. In contrast, in the cultured apocrine epithelial cells from axillary osmidrosis subjects, inhibition of JNK1 significantly reduced the expression of ApoD. Taken together, our study here revealed that increased JNK1 activation in the apocrine cells from axillary osmidrosis contributes to the increased ApoD expression, which in turn involved in the process of axillary osmidrosis.     

Characterization and expression of amphioxus ApoD gene encoding an archetype of vertebrate ApoD proteins

Here we report a homologue of the apolipoprotein D gene (AmphiApoD) in amphioxus, Branchiostoma belcheri tsingtauense, the first such finding in a basal chordate cephalochordate. The main features of the protein predicted from AmphiApoD are characteristic of the apolipoprotein D. Phylogenetic analysis places AmphiApoD at the base of the phylogenetic tree, suggesting that AmphiApoD is the archetype of the vertebrate ApoD genes. Both whole mount in situ hybridization and Northern blotting and RT-PCR as well as in situ hybridization histochemistry reveal that AmphiApoD is expressed in tissues derived from mesoderm and endoderm including notochord and hind-gut, which contrasts with the strong expression patterns of ApoD genes in the ectodermal derivatives in mammals and birds. The expression profiles of the ApoD gene may have been changed to be expressed in the endo-mesodermal derivatives in amphioxus after the vertebrate and cephalochordate lineages diverged; alternatively, the ApoD gene may first have been expressed in the endo-mesoderm during embryogenesis in the last common ancestor of all chordates, and subsequently came to be expressed in the ectodermal derivatives of vertebrates including mammals and birds.     

Loss of glial lazarillo, a homolog of apolipoprotein D, reduces lifespan and stress resistance in Drosophila

The vertebrate Apolipoprotein D (ApoD) is a lipocalin secreted from subsets of neurons and glia during neural development and aging . A strong correlation exists between ApoD overexpression and numerous nervous system pathologies as well as obesity, diabetes, and many forms of cancer . However, the exact relationship between the function of ApoD and the pathophysiology of these diseases is still unknown. We have generated loss-of-function Drosophila mutants for the Glial Lazarillo (GLaz) gene , a homolog of ApoD in the fruit fly, mainly expressed in subsets of adult glial cells. The absence of GLaz reduces the organism's resistance to oxidative stress and starvation and shortens male lifespan. The mutant flies exhibit a smaller body mass due to a lower amount of neutral lipids stored in the fat body. Apoptotic neural cell death increases in aged flies or upon paraquat treatment, which also impairs neural function as assessed by behavioral tests. The higher sensitivity to oxidative stress and starvation and the reduced fat storage revert to control levels when a GFP-GLaz fusion protein is expressed under the control of the GLaz natural promoter. Finally, GLaz mutants have a higher concentration of lipid peroxidation products, pointing to a lipid peroxidation protection or scavenging as the mechanism of action for this lipocalin. In agreement with Walker et al. (, in this issue of Current Biology), who analyze the effects of overexpressing GLaz, we conclude that GLaz has a protective role in stress situations and that its absence reduces lifespan and accelerates neurodegeneration.     

Structural insight into the dual ligand specificity and mode of high density lipoprotein association of apolipoprotein D

Human apolipoprotein D (ApoD) occurs in plasma associated with high density lipoprotein. Apart from the involvement in lipid metabolism, its binding activity for progesterone and arachidonic acid plays a role in cancer development and neurological diseases. The crystal structures of free ApoD and its complex with progesterone were determined at 1.8A resolution and reveal a lipocalin fold. The narrow, mainly uncharged pocket within the typical beta-barrel accommodates progesterone with its acetyl side chain oriented toward the bottom. The cavity adopts essentially the same shape in the absence of progesterone and allows complexation of arachidonic acid as another cognate ligand. Three of the four extended loops at the open end of the beta-barrel expose hydrophobic side chains, which is an unusual feature for lipocalins and probably effects association with the high density lipoprotein particle by mediating insertion into the lipid phase. This mechanism is in line with an unpaired Cys residue in the same surface region that can form a disulfide cross-link with apolipoprotein A-II.     

Cellular localization of apolipoprotein D and lecithin:cholesterol acyltransferase mRNA in rhesus monkey tissues by in situ hybridization

Apolipoprotein D (apoD) and lecithin:cholesterol acyl transferase (LCAT) are found on high density lipoprotein particles (HDLs) and have been postulated to form part of a complex involved in the transport of cholesterol from peripheral tissues to the liver for excretion. We have examined the sites of synthesis of the mRNAs for these two proteins in the rhesus monkey by in situ hybridization. ApoD mRNA-containing cells were widely distributed throughout peripheral tissues in interstitial and connective tissue fibroblasts often associated with blood vessels or capillaries. ApoD mRNA was also found localized in cells associated with peripheral nerves, neuroglial cells, cells in the subarachnoid space on the surface of the brain including the pial cells, perivascular cells, and scattered neurons in the brain. LCAT demonstrated a much more restricted pattern of synthesis and was found to be synthesized by hepatocytes, the basal cell layer of the epidermis, and in brain cell populations distinct from those that synthesize apoD. In the brain LCAT was synthesized by scattered neurons, neuroglial cells, ependymal cells, as well as a discrete cell layer in the cerebellum. ApoD has been shown to possess extensive homology to retinol binding protein, which has a binding pocket for vitamin A. We propose that apoD may also function to bind cholesterol or its derivatives in compartments not in direct contact with the blood. The findings of both apoD and LCAT synthesis in the brain suggest that they play a significant role in lipid transport in the brain.     

Apolipoprotein D

Apolipoprotein D (apoD) is a 29-kDa glycoprotein that is primarily associated with high density lipoproteins in human plasma. It is an atypical apolipoprotein and, based on its primary structure, apoD is predicted to be a member of the lipocalin family. Lipocalins adopt a beta-barrel tertiary structure and transport small hydrophobic ligands. Although apoD can bind cholesterol, progesterone, pregnenolone, bilirubin and arachidonic acid, it is unclear if any, or all of these, represent its physiological ligands. The apoD gene is expressed in many tissues, with high levels of expression in spleen, testes and brain. ApoD is present at high concentrations in the cyst fluid of women with gross cystic disease of the breast, a condition associated with increased risk of breast cancer. It also accumulates at sites of regenerating peripheral nerves and in the cerebrospinal fluid of patients with neurodegenerative conditions, such as Alzheimer's disease. ApoD may, therefore, participate in maintenance and repair within the central and peripheral nervous systems. While its role in metabolism has yet to be defined, apoD is likely to be a multi-ligand, multi-functional transporter. It could transport a ligand from one cell to another within an organ, scavenge a ligand within an organ for transport to the blood or could transport a ligand from the circulation to specific cells within a tissue.     

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease

ApoD (apolipoprotein D) is up-regulated in AD (Alzheimer's disease) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo, but the mechanism is unknown. Specific methionine residues may inhibit lipid peroxidation by reducing radical-propagating L-OOHs (lipid hydroperoxides) to non-reactive hydroxides via a reaction that generates MetSO (methionine sulfoxide). Since apoD has three conserved methionine residues (Met(49), Met(93) and Met(157)), we generated recombinant proteins with either one or all methionine residues replaced by alanine and assessed their capacity to reduce HpETEs (hydroperoxyeicosatetraenoic acids) to their HETE (hydroxyeicosatetraenoic acid) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the methionine residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated that Met(93) was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD methionine oxidation was associated with formation of apoD aggregates that were also detected in the hippocampus of AD patients. In conclusion, conversion of HpETE into HETE is mediated by apoD Met(93), a process that may contribute to apoD antioxidant function.     

Apolipoprotein D overexpression alters hepatic prostaglandin and omega fatty acid metabolism during the development of a non-inflammatory hepatic steatosis

Apolipoprotein D (ApoD) is a secreted lipocalin associated with neuroprotection and lipid metabolism. Overexpression of ApoD in mouse neural tissue induces the development of a non-inflammatory hepatic steatosis in 12-month-old transgenic animals. Previous data indicates that accumulation of arachidonic acid, ApoD's preferential ligand, and overactivation of PPARγ are likely the driving forces in the development of the pathology. However, the lack of inflammation under those conditions is surprising. Hence, we further investigated the apparent repression of inflammation during hepatic steatosis development in aging transgenic animals. The earliest modulation of lipid metabolism and inflammation occurred at 6 months with a transient overexpression of L-PGDS and concomitant overproduction of 15d-PGJ₂, a PPARγ agonist. Hepatic lipid accumulation was detectable as soon as 9 months. Inflammatory polarization balance varied in time, with a robust anti-inflammatory profile at 6 months coinciding with 15d-PGJ₂ overproduction. Omega-3 and omega-6 fatty acids were preferentially stored in the liver of 12-month-old transgenic mice and resulted in a higher omega-3/omega-6 ratio compared to wild type mice of the same age. Thus, inflammation seems to be controlled by several mechanisms in the liver of transgenic mice: first by an increase in 15d-PGJ₂ production and later by a beneficial omega-3/omega-6 ratio. PPARγ seems to play important roles in these processes. The accumulation of several omega fatty acids species in the transgenic mouse liver suggests that ApoD might bind to a broader range of fatty acids than previously thought.     

腋臭患者大汗腺中ApoD和AR的表达水平与气味强度间的关系

目的:明确大汗腺组织中ApoD和AR的表达水平与腋臭患者散发出气味间的关系,阐明两者在腋臭发病中的作用,为腋臭的临床治疗和干预奠定基础。方法:收集在我院整形外科就诊的腋臭病人腋下皮肤组织标本41例。手术前由3名医生共同判断患者腋下气味强度,按照能在≤1 m、≤3 m、≤5 m距离闻及气味把患者分为轻、中、重三组。随机抽取各组标本组织块进行RT-PCR检测,测定ApoD和AR在大汗腺组织中的含量。结果:根据气味强度判定标准,轻、中和重3组患者人数分别为:7人、11人和23人。按照AB 7500 Real-Time PCR系统分析结果所示ApoD相对表达量的等级与气味强度之间有线性关系(P0.05),ApoD相对表达量的等级随着气味强度的增加而升高;AR相对表达量的等级与气味强度之间有线性关系(P0.05),随着气味强度的增加而升高。结论:ApoD和AR的表达量与腋臭气味强度之间有着密切的联系。进一步证实AR可调节大汗腺组织ApoD的表达,为明确腋臭的发病机制提供了理论依据。     

Cellular Cholesterol Storage in the Niemann-Pick Disease Type C Mouse Is Associated with Increased Expression and Defective Processing of Apolipoprotein D

Abstract: Apolipoprotein D (apoD), a member of the lipocalin superfamily of ligand transporters, has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. We have previously established that apoD is a secreted protein from cultured mouse astrocytes and that treatment with the oxysterol 25-hydroxycholesterol markedly stimulates apoD release. Here, we have investigated expression and cellular processing of apoD in the Niemann-Pick type C (NPC) mouse, an animal model of human NPC, which is a genetic disorder affecting cellular cholesterol transport. NPC is phenotypically characterized by symptoms of chronic progressive neurodegeneration. ApoD gene expression was up-regulated in cultured NPC astrocytes and in NPC brain. ApoD protein levels were also increased in NPC brain with up to 30-fold higher apoD content in the NPC cerebellum compared with control mice. Subcellular fractionation of NPC brain homogenates revealed that most of the apoD was associated with the myelin fraction. ApoD was found to be a secreted protein from cultured normal astrocytes and treatment with the oxysterol, 25-hydroxycholesterol, markedly stimulated apoD release (by five- to 10-fold). By contrast, secretion of apoD from NPC astrocytes was markedly reduced and could not be stimulated by oxysterol treatment. Secretion of apoE, another apolipoprotein normally produced by astrocytes, was similar in NPC and control cells. Furthermore, apoE secretion was not potentiated by oxysterol treatment in either cell type. Plasma levels of apoD were sixfold higher in NPC, whereas hepatic levels were substantially reduced compared with controls, possibly reflecting reduced hepatic clearance of the circulating protein. These results reveal hitherto unrecognized defects in apoD metabolism in NPC that appear to be linked to the known defects in cholesterol homeostasis in this disorder.