Semliki Forest Virus Expression System Research Articles

Secretion from Bovine Chromaffin Cells Acutely Expressing Exogenous Proteins Using a Recombinant Semliki Forest Virus Containing an EGFP Reporter

Acute expression of recombinant proteins throughout a population of postmitotic bovine chromaffin cells was achieved using the Semliki Forest virus expression system (P. Liljestrom and H. Garoff (1991) Biotechnology 9:1356–1361). The virus was modified to express a green fluorescent protein, which faithfully reported the expression of the recombinant proteins. Two types of reporting virus were constructed: the first included a second subgenomic element, and the second an internal ribosome entry site. Both were used to express the recombinant proteins β-galactosidase, 5HT3 receptor, or tetanus toxin light chain. β-galactosidase was used to quantify the rate of expression of recombinant protein in chromaffin cells, the 5HT3 receptor to trigger secretion, and the toxin to block secretion. The experiments clearly show that infection and expression of recombinant proteins throughout a population of chromaffin cells do not, per se, affect the rate and extent of triggered exocytosis, endocytosis, or membrane recycling pathways. The catecholamine content of the cell is unaltered, and the secretory mechanism can be accessed within a few hours after infection. This noncytopathic method of acutely expressing specific proteins at physiological levels in chromaffin cells offers a powerful new tool for dissecting the roles of many proteins implicated in exo- and endocytosis.

Generation of monoclonal antibodies to native human immunodeficiency virus type 1 envelope glycoprotein by immunization of mice with naked RNA

The Semliki Forest virus (SFV) vector system is a new approach for in vivo expression of heterologous proteins and can also be used to generate specific immune responses in animal models. HIV-1 envelope glycoprotein produced using the SFV expression system is correctly folded, cleaved, transported to the cell surface and exhibits functional activity. We evaluated a recombinant Semliki Forest virus naked RNA-based immunization protocol for generation of monoclonal antibodies against the HIV-1 envelope glycoprotein. In vitro-transcribed RNA encoding for the SFV replicase complex and Env protein of HIV-1 (HXB2 strain) was injected intramuscularly to mice. This approach elicited an Env-specific antibody response in four mice out of five and a monoclonal antibody, 12H2, directed against gp41 was produced. Our results show that recombinant SFV RNA immunization can potentially be used as a quick and direct method to produce monoclonal antibodies, with the particular advantage that vectored RNA, rather than purified antigen, delivers a complex oligomer produced correctly.

Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice

The replicon of Semliki Forest virus (SFV) offers the possibility to direct high-level, transient expression of heterologous proteins in vivo. We initiated studies to determine the possibility of employing the SFV expression system for recombinant vaccine purposes. Mice immunized with recombinant SFV encoding Influenza A nucleoprotein (NP) or E. coli LacZ developed long-lasting antigen-specific IgG levels and induction of cytotoxic T-cell (CTL) memory that persisted for over one year. Predominantly type 1 T-helper cells were induced as shown by IgG subclass ELISA. Humoral and cell-mediated immune responses could be induced upon delivery by several administration routes and mucosal immunizations induced secretory IgA in the respiratory tract. Development of immune responses against the vector itself did not inhibit boost responses by subsequent immunizations with recombinant SFV. Immunization of mice with vectors encoding the Influenza A virus antigens nucleoprotein (NP) and hemagglutinin (HA) resulted in immune responses that were protective against challenge infection with Influenza virus.

Extracts and Constituents of Hypericum Perforatum Inhibit the Binding of Various Ligands to Recombinant Receptors Expressed with the Semliki Forest Virus System

Extracts, fractions and constituents of Hypericum perforatum were studied for in vitro receptor binding with various ligands to recombinant CNS receptors expressed with the Semliki Forest virus expression system. For this purpose we have prepared membranes of CHO cells with high density of several opioid, serotonin, estrogen, histamine, GABAA, neurokinin and metabotropic glutamate receptors, respectively. A lipophilic Hypericum fraction revealed relatively potent inhibition to the binding of the μ-, δ- and -opioid and the 5-HT6 and 5-HT7 receptors. Moreover, Hypericum constituents such as the naphthodianthrones, hypericin and pseudohypericin, and the phloroglucinole hyperforin inhibited both binding to the opioid and serotonin receptors in the lower micromolar range. Estrogen binding was 50% inhibited by the biflavonoid I3,II8-biapigenin at micromolar concentration. The lipophilic Hypericum fraction provided a less potent inhibition of the neurokinin-1 receptor binding compared to the opioid and serotonin receptors. A total ethanolic Hypericum extract potently inhibited GABAA binding at approximately 3 μg/ml. This inhibition is however not specific to Hypericum, since extracts of plants like Valeriana officinalis and Passiflora incarnata showed similar inhibitions. Binding to neither histamine nor metabotropic glutamate receptors was affected by Hypericum extracts. These results support the hypothesis that several active constituents of Hypericum might in a synergistic way contribute to its antidepressant effect in the central nervous system.

Expression of the α1b-Adrenergic Receptor and G Protein Subunits in Mammalian Cell Lines Using the Semliki Forest Virus Expression System

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the α1b-adrenergic receptor (AR) alone or in the presence of the G protein αq and/or β2 and γ2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-α1b-AR particles resulted in high specific binding activity of the α1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours postinfection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Soluble expression and complex formation of proteins required for HCMV DNA replication using the SFV expression system.

Several of the viral proteins required for human cytomegalovirus (HCMV) DNA replication have been difficult to study due to their low abundance in infected cells and low solubility in bacterial or insect-cell expression systems. Therefore we used the Semliki Forest virus expression system to express these proteins in mammalian cells. All of the recombinant proteins were soluble, on the basis of ultracentrifugation properties and their ability to be immunoprecipitated from solution with specific antibodies. Pulse-chase analysis of the 86-kDa major immediate-early protein (IE86) revealed two expressed forms-a precursor and a product-indicating that this recombinant protein, like the native HCMV protein, is posttranslationally processed. The recombinant proteins (polymerase core and accessory as well as the IE86 and pUL84) formed stable complexes similar to those known to form in HCMV-infected cells. The recombinant DNA polymerase holoenzyme also exhibited enzyme activity that was phosphonoformic acid sensitive, as is the infected-cell DNA polymerase activity. This expression system offers many advantages for the expression and study of the HCMV replication proteins, including the expression of soluble, active proteins that are able to interact to form complexes. Additionally, the relative ease with which SFV recombinants can be made lends itself to the construction and evaluation of mutants.

Hypericum perforatum inhibits the binding of mu- and kappa-opioid receptor expressed with the Semliki Forest virus system.

The effects of Hypericum perforatum extracts on in vitro [3H]naloxone binding to the human mu- and rat kappa-opioid receptors were studied in chinese hamster ovary (CHO) cells using the Semliki Forest virus (SFV) expression system. Binding of [3H]naloxone to the mu- and kappa-opioid receptor was inhibited in the presence of Hypericum extracts showing IC50 values of approximately 25 and 90 micrograms/ml, respectively. In contrast, extracts of Valeriana officinalis did not inhibit binding to the mu-opioid receptor. Also, single constituents of H. perforatum like the flavonoids quercetin and kaempferol and the glycosilated flavonoid quercitrin did not inhibit [3H]naloxone binding to the mu-opioid receptor up to a concentration of 10 microM. The present in vitro data may suggest a new possible mechanism for the anti-depressant effect of H. perforatum.

Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors.

Efficient and controllable expression of a transgene usually requires the presence of intron sequences and much efforts have been made to produce retrovirus vectors that can transduce and integrate genes with introns. However, this has proven difficult because the viral RNA is spliced when it is synthesized in the nucleus of a producer cell. We describe a novel approach to avoid this problem. In our system the retroviral RNA is synthesized in the cytoplasm of the cell, not in the nucleus, in a reaction driven by the Semliki Forest virus (SFV) expression system. The approach was tested with a recombinant Moloney murine leukemia virus genome containing the chloramphenicol acetyltransferase (CAT) gene in association with an intron. This was inserted into a SFV transcription plasmid and the corresponding SFV vector RNA was transcribed in vitro. BHK-21 cells were then transfected with this vector RNA together with two additional SFV vectors that encode the Moloney murine leukemia virus packaging proteins. Retrovirus vectors containing intron-CAT sequences were produced at titers up to 1.3 x 10(6) infectious particles per ml during a 5-hr incubation period. The vectors faithfully transduced the intron-containing CAT gene into NIH 3T3 cells, where the intron-CAT RNA was subjected to efficient splicing and used for high level enzyme expression. Thus, the results show that intron containing genes can be efficiently packaged into retrovirus vectors by the SFV expression system.

Differential Effects of the Swedish Mutant Amyloid Precursor Protein on β-Amyloid Accumulation and Secretion in Neurons and Nonneuronal Cells

Expression of the Swedish DeltaNL mutation in the beta-amyloid precursor protein (APPDeltaNL) dramatically increases Abeta generation in nonneuronal cell lines, although it is unclear whether intracellular levels of beta-amyloid (Abeta) are also elevated after APPDeltaNL expression. Furthermore, the effects of expressing APPDeltaNL in neurons on the production and secretion of Abeta-(1-40) and Abeta-(1-42) are unknown. To address these issues, we examined the generation of both intracellular and secreted Abeta-(1-40) and Abeta-(1-42) in human neuronal NT2N cells, in primary rat astrocytes, and in Chinese hamster ovary cells engineered to express wild-type APP or APPDeltaNL using a recombinant Semliki Forest virus expression system. Expression of APPDeltaNL led to a marked increase in APPbeta and the C-terminal fragment containing the entire Abeta sequence (C99) in all cells tested. However, a dramatic elevation of intracellular and secreted Abeta-(1-40) and Abeta-(1-42) was seen only in astrocytes and Chinese hamster ovary cells. The DeltaNL mutation did not cause a significant increase in intracellular or secreted Abeta-(1-40) or Abeta-(1-42) in NT2N cells. Since NT2N cells expressing APPDeltaNL accumulate much higher levels of C99 than cells expressing wild-type APP, we conclude that the rate-limiting step in Abeta production could be the further processing of C99 by gamma-secretase in these cells. These results show that the Swedish DeltaNL mutation causes nonneuronal cells to process APP via pathways more in common with the metabolism of wild-type APP in neurons.

Interaction of nascent ApoE2, ApoE3, and ApoE4 isoforms expressed in mammalian cells with amyloid peptide beta (1-40). Relevance to Alzheimer's disease.

Population studies have established that one of the common isoforms of apolipoprotein E, the apoE4, is associated with higher incidence and earlier age of onset of late onset familial Alzheimer's disease (AD), whereas apoE2 may have the opposite effect. The apoE3 and apoE4 isoforms were shown to display different binding reactivities with amyloid beta peptide (Abeta) and tau protein in vitro. On the basis of these findings, it has been proposed that the apoE isoforms may modulate positively or negatively the formation of either the neurofibrillary tangles or the amyloid deposits in the brain of patients with AD. To study the interaction of Abeta with nascent apoE isoforms we have expressed their cDNAs in baby hamster kidney (BHK-21) cells using the Semliki Forest Virus expression system. Analysis of the secreted apoE by one- and two-dimensional gel electrophoresis and immunoblotting showed that the nascent apoE is heavily modified with carbohydrate chains containing sialic acid. A dimeric form of apoE is formed with apoE2 and apoE3 but not with apoE4 isoforms. Analysis of the interaction of nascent apoE2, apoE3, and apoE4 produced by BHK-21 cells with Abeta (1-40) under physiological conditions (pH 7.4, 37 degrees C) showed that the efficiency of the apoE monomer-Abeta complex formation follows the order apoE2 > apoE3 >> apoE4. In addition, the apoE2 dimer formed a complex with Abeta more efficiently than the apoE3 dimer. The isoform-specific differences in binding were temperature-dependent and are attenuated upon decrease of the temperature. The binding behavior of the monomeric apoE is different from that reported for plasma apoE3 and apoE4 or commercially available apoE3 and apoE4 preparations and similar to that described for apoE3 and apoE4 produced by human embryonic kidney (HEK-293) cells. It appears that the efficiency of binding between each of three main apoE isoforms and Abeta correlates inversely with the risk of developing late-onset familial AD and may indicate possible involvement of apoE in the binding and clearance of Abeta in vivo.

Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus.

GP4 is a minor structural glycoprotein encoded by ORF4 of Lelystad virus (LV). When it was immunoprecipitated from cell lysates and extracellular virus of CL2621 cells infected with LV, it was shown to have an apparent molecular mass of approximately 28 and 31 kDa, respectively. This difference in size occurred because its core N-glycans were modified to complex type N-glycans during the transport of the protein through the endoplasmic reticulum and Golgi compartment. A panel of 15 neutralizing monoclonal antibodies (MAbs) reacted with the native GP4 protein expressed by LV and the recombinant GP4 protein expressed in a Semliki Forest virus expression system. However, these MAbs did not react with the GP4 protein of U.S. isolate VR2332. To map the binding site of the MAbs, chimeric constructs composed of ORF4 of LV and VR2332 were generated. The reactivity of these constructs indicated that all the MAbs were directed against a region spanning amino acids 40 to 79 of the GP4 protein of LV. Six MAbs reacted with solid-phase synthetic dodecapeptides. The core of this site consists of amino acids 59 to 67 (SAAQEKISF). Comparison of the amino acid sequences of GP4 proteins from various European and North American isolates indicated that the neutralization domain spanning amino acids 40 to 79 is the most variable region of GP4. The neutralization domain of GP4, described here, is the first identified for LV.

High level expression of the human 5-HT1D alpha serotonin receptor using the Semliki Forest Virus expression system.

Annals of the New York Academy of SciencesVolume 812, Issue 1 p. 229-230 High Level Expression of the Human 5-HT1Dα Serotonin Receptor Using the Semliki Forest Virus Expression System J. STABLES, J. STABLES Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this authorS. REES, S. REES Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this authorK. LUNDSTROM, K. LUNDSTROM Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this authorD. LIVINGSTONE, D. LIVINGSTONE Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this authorM. G. LEE, M. G. LEE Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this author J. STABLES, J. STABLES Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this authorS. REES, S. REES Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this authorK. LUNDSTROM, K. LUNDSTROM Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this authorD. LIVINGSTONE, D. LIVINGSTONE Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this authorM. G. LEE, M. G. LEE Biology Division Glaxo-Wellcome Gunnels Wood Road Stevenage SG1 2NY, United KingdomSearch for more papers by this author First published: 17 December 2006 https://doi.org/10.1111/j.1749-6632.1997.tb48185.xCitations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume812, Issue1Receptor Classification: The Integration of Operational, Structural, and Transductional InformationMay 1997Pages 229-230 RelatedInformation

Binding of [125I] AB‐MECA to the human cloned adenosine A3 receptor using the semliki forest virus expression system

Binding of [125I] AB‐MECA to the human cloned adenosine A3 receptor using the semliki forest virus expression system

Expression of Ligand-Gated Ion Channels with the Semliki Forest Virus Expression System

Two types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system.The cDNAs for mouse serotonin 5-HT3 receptor and rat and human purinoreceptor P2x subtypes were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper2 RNA into BHK cells, where in vivo packaging resulted in high titer SFV-5-HT3 and SFV-P2x virus stocks. Infection of BHK, CHO and RJN cells resulted in high-level expression of recombinant receptors. Saturation binding analysis indicated the presence of more than 3 × 106 5-HT3 receptors per cell. Binding studies on isolated membranes yielded from 10 to 60 pmol of either 5-HT3 or P2x receptor per mg protein. Functional responses to the P2x receptors were demonstrated in SFV-infected CHO cells by Ca2+ mobilization or by 45Ca2+ influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT3 and SFV-P2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purification procedure of SFV-expressed recombinant receptors a histidine tag was introduced at the C-terminus of the 5-HT3 receptor. This 5-HT3His receptor showed high levels of expression, specific binding and high amplitude electrophysiological responses. For large scale expression the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5 liter fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 × 106 receptors per cell.

Binding of [125I] AB-MECA to the human cloned adenosine A3 receptor using the semliki forest virus expression system

The cDNA for the human adenosine A3 receptor was introduced into the pSFV1 vector, and the in vitro transcribed RNA was electroporated into baby hamster kidney (BHK) cells with pSFV-Helper RNA. This protocol resulted in packaging of a high titre Semliki Forest Virus (SFV)-A3 virus stock. Infection of confluent Chinese hamster ovary (CHO) cells with the SFV-A3 virus stock resulted in an expression of human adenosine A3 receptors that was twofold more than that obtained with usual transfection methods (as determined by radioligand binding studies). The binding of [125I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyl-uronamide ([125I]AB-MECA) was specific and saturable (pKd = 8.8; Bmax = 0.5 pmol mg−1 protein). Adenosine receptor ligands were evaluated for their binding afffinities at the human cloned adenosine A3 receptor. The rank order of affinities of the ligands were: CGS 15943 > IB-MECA > APNEA > ligands with selectivity for adenosine A1, A2A, and A2B, receptors. However, the A1 selective ligand, GR79236 had little or no affinity for the human adenosine A3 receptor. In conclusion, the SFV expression system can be used to express the human cloned adenosine A3 receptor at high levels in CHO cells. This study has examined the binding affinities at the human cloned adenosine A3 receptor, of an extensive range of ligands for the adenosine family of receptors. Furthermore, CGS 15943 has been identified as a ligand with high affinity at the human cloned adenosine A3 receptor. Drug Dev. Res. 40:35–40, 1997. © 1997 Wiley-Liss, Inc.

Human Papillomavirus Type 16 Capsid Proteins Produced from Recombinant Semliki Forest Virus Assemble into Virus-like Particles

Several neutralizing sites of the human papillomavirus (HPV) capsid are known to be critically dependent on the conformation of the capsid. However, efficient production of HPV16 capsids in mammalian cells has been difficult, possibly because the HPV genome contains negative regulatory elements. To circumvent these problems, we cloned the HPV16 L1 and L2 genes from a healthy HPV16-infected woman into a Semliki Forest virus based expression vector (P. Liljeström and H. Garoff,Biotechnology9, 1356–1361, 1991). Recombinant HPV16 L1- or L2-producing Semliki Forest virus was generated and used for infection of mammalian cells. The HPV16 L1 and L2 proteins were efficiently expressed and the majority of the L1 protein self assembled into virus-like particles (VLPs). Coexpression of L1 and L2 resulted in incorporation of L2 into the VLPs. The particles had a density of ∼1.3 g/ml as determined by density gradient centrifugation. Transmission electron microscopy revealed that the particles had a morphology similar to native virions. The HPV16 VLPs produced by the Semliki Forest virus expression system may be useful as a conformationally correctly assembled target for studies of HPV attachment, assembly, serology, or vaccination.

Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture.

Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface-exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role.

De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.

Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae. Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein. Thy1, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.

Production of Recombinant PSA and HK2 and Analysis of Their Immunological Cross-Reactivity

Measurements of prostate-specific antigen (PSA) in serum are widely used to monitor patients with prostate cancer, but the attenuation of the assay response by PSA complexed to protease inhibitors has been shown to affect the results in certain assay designs. Moreover, the human glandular kallikrein-2 (hK2), a kallikrein-like serine protease that is 80% similar to PSA, might interfere with the specific detection of PSA by immunological cross-reactivity. We have expressed hK2 and PSA in eucaryotic cells using the Semliki Forest Virus expression system and studied the reactivity of 18 monoclonal anti-PSA IgGs. Five of them cross-reacted with identical affinities to recombinant hK2 whereas 13 recognized PSA alone. The antibodies that recognized both PSA and hK2 bind to a region of the protein that is exposed when PSA is complexed to alpha-1-antichymotrypsin.

Intracellular routing of human amyloid protein precursor: Axonal delivery followed by transport to the dendrites

A characteristic neuropathological feature of Alzheimer's disease is the cerebral deposition of amyloid plaques. These deposits contain beta A4 amyloid peptide, a cleavage product of the transmembrane protein amyloid protein precursor (APP). Despite numerous studies on the processing of the different APP isoforms in non-neuronal cells, little is known about its sorting and transport in neurons of the central nervous system (CNS). To analyze this question we expressed in cultured rat hippocampal neurons the human APP 695, tagged at its N-terminus with the myc epitope, using the Semliki forest virus (SFV) expression system. APP was first delivered from the cell body to the axon and later appeared also in the dendrites. Inhibition of protein synthesis at the time of axonal expression did not block the late appearance of the protein in the dendrites. An antibody directed against the myc tag, bound to the cell surface at 4 degrees C at the time of axonal APP expression, could be chased to the dendritic domain after subsequent incubation at 37 degrees C. These results suggest that the newly synthesized APP, after initial axonal delivery, may be transported to the dendrites by a transcytotic mechanism. The routing of APP in polarized neurons is different from that of polarized epithelial cells, in which the protein is delivered basolaterally, arguing for neuronal specific sorting and processing mechanisms.