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#26826316   2016/03/22 Save this To Up

Production of a soluble and functional recombinant apolipoproteinD in the Pichia pastoris expression system.

ApolipoproteinD (ApoD) is a human glycoprotein from the lipocalin family. ApoD contains a conserved central motif of an 8-stranded antiparallel β-sheet, which forms a beta-barrel that can be used for transport and storage of diverse hydrophobic ligands. Due to hydrophobic nature of ApoD, it has been difficult to generate a recombinant version of this protein. In the present work, we aimed at the production of ApoD in the robust Pichia pastoris expression system. To this end, the ApoD gene sequence was synthesized and subcloned for expression in the yeast host cells. Following integration of the ApoD gene into the yeast genomic region using homologous recombination, the ApoD recombinant protein was induced using methanol, reaching its maximum induction at 96 h. Having purified the ApoD recombinant protein by affinity chromatography, we measured the dissociation constant (KD) using its natural ligands: progesterone and arachidonic acid. Our results provide a viable solution to the production of recombinant ApoD protein in lieu of previous obstacles in generating soluble and functional ApoD protein.

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#23777559   2013/07/29 Save this To Up

Lipid-binding properties of human ApoD and Lazarillo-related lipocalins: functional implications for cell differentiation.

Lipocalins are a family of proteins characterized by a conserved eight-stranded β-barrel structure with a ligand-binding pocket. They perform a wide range of biological functions and this functional multiplicity must relate to the lipid partner involved. Apolipoprotein D (ApoD) and its insect homologues, Lazarillo (Laz) and neural Lazarillo (NLaz), share common ancestral functions like longevity, stress resistance and lipid metabolism regulation, coexisting with very specialized functions, like courtship behavior. Using tryptophan fluorescence titration, we screened the binding of 15 potential lipid partners for NLaz, ApoD and Laz and uncovered several novel ligands with apparent dissociation constants in the low micromolar range. Retinoic acid (RA), retinol, fatty acids and sphingomyelin are shared ligands. Sterols, however, showed a species-specific binding pattern: cholesterol did not show strong binding to human ApoD, whereas NLaz and Laz did bind ergosterol. Among the lipocalin-specific ligands, we found that ApoD selectively binds the endocannabinoid anandamide but not 2-acylglycerol, and that NLaz binds the pheromone 7-tricosene, but not 7,11-heptacosadiene or 11-cis-vaccenyl acetate. To test the functional relevance of lipocalin ligand binding at the cellular level, we analyzed the effect of ApoD, Laz and NLaz preloaded with RA on neuronal differentiation. Our results show that ApoD is necessary and sufficient to allow for RA differentiating activity. Both human ApoD and Drosophila NLaz successfully deliver RA to immature neurons, driving neurite outgrowth. We conclude that ApoD, NLaz and Laz bind selectively to a different but overlapping set of lipid ligands. This multispecificity can explain their varied physiological functions.

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#22398376   2012/05/22 Save this To Up

Amyloid-β25-35 induces apolipoprotein D Synthesis and growth arrest in HT22 hippocampal cells.

Apolipoprotein D (ApoD) is a secreted glycoprotein that is markedly induced in several pathological and stressful conditions in the nervous system. In the central nervous system, ApoD expression is upregulated during aging, after traumatic brain injury, and in several human neuropathologies such as Alzheimer's disease (AD), where it is found associated with amyloid-β (Aβ) plaques. Recent studies have indicated that ApoD has an important function as a neuroprotective and antioxidant protein. The aim of this work is to study the effect of the peptide fragment Aβ25-35, which is believed to play a major role in the neurodegenerative process of AD, in ApoD expression in a mouse hippocampal cell line. In addition, we studied whether direct addition of exogenous human recombinant ApoD protein has neuroprotective effect against Aβ25-35 treatment on neuronal cells. Our results demonstrate that Aβ25-35 induces ApoD expression in hippocampal cells in response to stress-induced growth arrest. This observed relationship between Aβ and ApoD expression could explain the elevated levels of ApoD found in AD brain, where it may be a neuroprotective molecule in the course of AD, probably related to its lipid transport function or a direct antioxidant property. However, the addition of exogenous human recombinant ApoD does not exert any protective effect, most likely due to its major structural modifications.

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#22150111   2012/04/12 Save this To Up

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.

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#19001418   2009/01/05 Save this To Up

p53 family members regulate the expression of the apolipoprotein D gene.

p73 and p63 are members of the p53 gene family that play an important role in development and homeostasis, mainly by regulating transcription of a variety of genes. We report here that apolipoprotein D (apoD), a member of the lipocalin superfamily of lipid transport proteins, is a direct transcriptional target of the p53 family member genes. We found that the expression of apoD was specifically up-regulated by either TAp73 or TAp63 but not significantly by p53. In addition, apoD transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abolishes induction of apoD transcription following cisplatin treatment. We also identified a p73/p63-binding site in the promoter of the apoD gene that is responsive to the p53 family members. The ectopic expression of TAp73 as well as the addition of recombinant human apoD to culture medium induced the osteoblastic differentiation of the human osteosarcoma cell line Saos-2, as assessed by alkaline phosphatase activity. Importantly, apoD knockdown abrogated p73-mediated alkaline phosphatase induction. Moreover, TAp73-mediated apoD expression was able to induce morphological differentiation, as well as expression of neuronal markers, in the human neuroblastoma cell line SH-SY5Y. These results suggest that apoD induction may mediate the activity of p73 in normal development.

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#16322568   2005/12/23 Save this To Up

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.

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#15192024   2004/07/23 Save this To Up

Apolipoprotein D and platelet-derived growth factor-BB synergism mediates vascular smooth muscle cell migration.

We identified apolipoprotein (apo)D in a search for proteins upregulated in a posttranscriptional manner similar to fibronectin in motile smooth muscle cells (SMCs). To address the function of apoD in SMCs, we cloned a partial apoD cDNA from ovine aortic (Ao) SMCs using RT-PCR. We documented a 2.5-fold increase in apoD protein but no increase in apoD mRNA in Ao SMCs 48 hours after a multiwound migration assay (P<0.01). Confocal microscopy revealed prominent perinuclear and trailing edge expression of apoD in migrating SMCs but not in the confluent monolayer. Stimulation of Ao SMCs with 10 ng/mL platelet-derived growth factor (PDGF)-BB increased apoD protein expression (P<0.05). Moreover, PDGF-BB-stimulated migration of human pulmonary artery SMCs was suppressed by knock-down of apoD using RNAi. Stable overexpression of apoD in Ao SMCs cultured in 10% fetal bovine serum promoted random migration by 62% compared with vector-transfected cells (P<0.01). Overexpression of apoD or addition of exogenous apoD to a rat aortic SMC line (A10) stimulated their migration in response to a subthreshold dose of PDGF-BB (P<0.05). This was unrelated to increased phosphorylation of ERK1/2 or of phospholipase C-gamma1, but correlated with enhanced Rac1 activation. This study shows that apoD can be expressed or taken up by SMCs and can regulate their motility in response to growth factors.

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#11833713   2002/02/08 Save this To Up

Structure-function relationships of human apolipoprotein D an immunochemical analysis.

Apolipoprotein D (apoD), a 169 amino acid member of the lipocalin family, is thought to be a transporter of small, hydrophobic ligands. A panel of 10 anti-apoD monoclonal antibodies (mAbs) was prepared and characterized in order to define apoD structure-function relationships. An apoD epitope map was constructed based on reactivity of the mAbs with apoD fragments. Three mAbs react with epitopes between apoD residues 7-78, seven mAbs with epitopes between residues 128-169, one mAb recognizes an epitope that straddles residues 99-102 and one mAb is specific for an epitope composed of non-contiguous apoD residues. Several pairs of mAbs whose respective epitopes are widely separated in apoD primary structure can compete for binding to immobilized apoD. This would be consistent with the compact beta-barrel tertiary structure that apoD is thought to adopt. None of the mAbs block the interaction of apoD with pregnenolone, a putative physiological ligand for apoD.

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#11180564   2001/02/22 Save this To Up

Bacterially produced apolipoprotein D binds progesterone and arachidonic acid, but not bilirubin or E-3M2H.

Apolipoprotein D (ApoD) constitutes an atypical lipoprotein in so far as it is predominantly found associated with HDL particles but belongs to the lipocalin structural family. Apart from its involvement in serum lipid transport it is abundant in various tissues, and differing physiological functions have been ascribed to it. We have now developed an E. coli expression system that permits the efficient production of biochemically homogeneous ApoD via secretion into the bacterial periplasm. Detailed ligand binding studies by fluorescence titration revealed that progesterone and arachidonic acid are complexed with dissociation constants both in the 1 microM range, whereas the presumed ligands pregnenolone, bilirubin and E-3M2H are not recognized by the recombinant protein. In contrast with previous reports it thus appears that ApoD discriminates well in its binding function between closely related compounds.

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#10820003   2000/07/06 Save this To Up

Mapping the CD4 binding domain of gp17, a glycoprotein secreted from seminal vesicles and breast carcinomas.

gp17, a secretory CD4-binding factor isolated from the human seminal plasma, is identical to the gross cystic disease fluid protein-15, a specific marker for primary and metastatic breast tumors. We previously demonstrated that gp17 binds to CD4 with high affinity and strongly inhibits T lymphocyte apoptosis induced by sequential cross-linking of CD4 and T cell receptor (TCR). To further characterize the gp17/CD4 interaction and map the gp17 binding site, we produced a secreted form of recombinant gp17 fused to human IgG1 Fc, gp17-Ig. We showed that gp17-Ig exhibits a binding affinity for CD4 similar to that of native gp17. As no information about gp17 structure is presently available, 99 overlapping gp17 peptides were synthesized by the Spot method, which allowed the mapping of two CD4 binding regions. Alanine scanning of CD4-reactive peptides identified critical residues, selected for site-directed mutagenesis. Nine gp17-Ig mutants were generated and characterized. Three residues within the carboxy-terminal region were identified as the major binding domain to CD4. The Spot method combined with mutagenesis represents a refined approach to distinguish the contact residues from the ones contributing to the conformation of the CD4-binding domain.

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