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R Cappai

Publications and source records attributed to R Cappai.

53 records · Page 3Linked to original sources
PubMed1998

Effects of the amyloid protein precursor of Alzheimer's disease and other ligands of the LDL receptor-related protein on neurite outgrowth from sympathetic neurons in culture.

The amyloid protein precursor (APP) of Alzheimer's disease can stimulate neurite outgrowth in vitro. The receptor responsible for this effect has not been identified. Kunitz protease inhibitor (KPI)-containing forms of APP bind to the low-density lipoprotein receptor-related protein (LRP). As LRP may regulate neurite outgrowth, we examined whether the effects of APP are mediated by LRP. Inhibitors of LRP decreased neurite outgrowth from chick sympathetic neurons. Most LRP ligands (alpha2-macroglobulin, lactoferrin, and lipoprotein lipase) stimulated outgrowth. However, in soluble form, the KPI-containing APP751 was a weak inhibitor of outgrowth. In substrate-bound form, both APP751 and APP695 (which does not bind to LRP) stimulated outgrowth. Thus the effect of substrate-bound APP on neurite outgrowth is not mediated by LRP.

Alzheimer Disease↗
PubMed1998

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein.

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.

Adenosine Diphosphate↗
PubMed1998

Subcellular localization of the Alzheimer's disease amyloid precursor protein and derived polypeptides expressed in a recombinant yeast system.

Different isoforms and derived polypeptides of the Alzheimer's disease amyloid protein precursor (A beta PP) have been expressed in the yeast Pichia pastoris. The expression characteristics of the different A beta PP polypeptides were studied by post-embedding immunogold electron microscopy with various A beta PP antibodies. The site of intracellular expression could be readily identified with specific antibodies. Full length A beta PP was expressed in association with the nuclear membrane and the endoplasmic reticulum. Secretory derivatives of A beta PP were localized in membrane-bound secretory vesicles. A construct encoding two copies of beta A4[1-42] linked head-to-tail (beta A4duplex) accumulated as irregular dense cytoplasmic and intranuclear inclusions which reacted with all beta A4 antibodies tested. A beta A4-C-terminal construct accumulated into membranous structures in the cytoplasm and nucleus and reacted with most antibodies to beta A4 and the cytoplasmic domain of A beta PP. The two shorter constructs containing the beta A4 sequence formed similar intranuclear aggregates to those reported for intranuclear inclusions of polyglutamine peptides from huntingtin (in Huntington's disease) and ataxin protein fragments (in spinocerebellar ataxia). This is of interest because intracellular aggregation of the polyglutamine and beta A4 peptides may affect cells by similar toxic mechanisms. These studies demonstrate clear differences in the expression properties of different A beta PP polypeptides.

Alzheimer Disease↗
PubMed1997

Expression and analysis of heparin-binding regions of the amyloid precursor protein of Alzheimer's disease.

Deletion mutagenesis studies have suggested that there are two domains within APP which bind heparan sulphate. These domains have been cloned and expressed in the yeast Pichia pastoris. Both recombinant proteins bound to heparin. One domain (APP316-447) was further characterised by binding studies with peptides encompassing this region. Peptides homologous to APP316-346 and APP416-447 were found to bind heparin. Circular dichroism studies show that APP416-447 shifted towards an alpha-helical conformation in the presence of heparin. This study suggests that heparin-binding domains may lie within regions high in alpha-helical structure.

Alzheimer Disease↗
PubMed1997

Alzheimer's disease-associated presenilin 1 in neuronal cells: evidence for localization to the endoplasmic reticulum-Golgi intermediate compartment.

The recently identified Alzheimer's disease-associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane-spanning proteins. Rabbit antibodies to the N-terminal domain of PS1 detected PS1 in human neuroblastoma SH-SY5Y wild type and PS1 transfectants (SY5Y-PS1) as well as in mouse P19, in CHO-K1 and CHO-APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full-length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) marker protein ERGIC-53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16 degrees C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC-53 with PS1 in the SY5Y-PS1 cells. PS1 labeling of SY5Y-PS1 and P19 cells showed overlap of the cis-Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y-PS1 cells and CHO-APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments.

Alzheimer Disease↗
PubMed1997

Expression of human amyloid precursor protein ectodomains in Pichia pastoris: analysis of culture conditions, purification, and characterization.

We have examined the use of the yeast Pichia pastoris for expression of the human amyloid precursor protein (APP). The ectodomains of the isoforms APP695, APP751, and APP770 were expressed in both P. pastoris protease-deficient strain SMD1163 and wild-type strain GS115, using the secretion vector pHIL-S1. Expression of recombinant APP in each of these strains produced intact recombinant protein, together with a small number of breakdown products. The levels of these breakdown products were not significantly altered by expression in the protease-deficient strain compared with wild-type GS115. The effects of induction time and medium composition on recombinant APP stability were also examined. After optimization of expression and culture conditions, baffled shaker flask cultures of clones selected for high expression routinely yielded 13-24 mg/liter recombinant protein following a two-step purification procedure. The recombinant isoforms possessed the heparin binding, metal binding, and Kunitz-type protease inhibitor properties of human brain-derived APP. These data indicate that P. pastoris is an appropriate laboratory-scale expression system for production of sufficient quantities of recombinant APP for use in biological studies.

Amyloid beta-Protein Precursor↗
PubMed1997

Identification of heparin-binding domains in the amyloid precursor protein of Alzheimer's disease by deletion mutagenesis and peptide mapping.

Recent studies have shown that the binding of the amyloid protein precursor (APP) of Alzheimer's disease to heparan sulfate proteoglycans (HSPGs) can modulate a neurite outgrowth-promoting function associated with APP. We used three different approaches to identify heparin-binding domains in APP. First, as heparin-binding domains are likely to be within highly folded regions of proteins, we analyzed the secondary structure of APP using several predictive algorithms. This analysis showed that two regions of APP695 contain a high degree of secondary structure, and clusters of basic residues, considered mandatory for heparin binding, were found, principally within these regions. To determine which domains of APP bind heparin, deletion mutants of APP695 were prepared and analyzed for binding to a heparin affinity column. The results suggested that there must be at least two distinct heparin-binding regions in APP. To identify novel heparin-binding regions, peptides homologous to candidate heparin-binding domains were analyzed for their ability to bind heparin. These experiments suggested that APP contains at least four heparin-binding domains. The presence of more than one heparin-binding domain on APP suggests the possibility that APP may interact with more than one type of glycosaminoglycan.

Alzheimer Disease↗
PubMed1996

Secreted glypican binds to the amyloid precursor protein of Alzheimer's disease (APP) and inhibits APP-induced neurite outgrowth.

The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican bound strongly to the APP affinity column. Purified glypican bound to APP with an equilibrium dissociation constant of 2.8 nM and inhibited APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican was specific for APP, as glypican did not inhibit laminin-induced neurite outgrowth. Furthermore, treatment of cultures with 4-methylumbelliferyl-beta-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibited APP-induced neurite outgrowth but did not inhibit laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP.

Alzheimer Disease↗
PubMed1995

Candidate gamma-secretases in the generation of the carboxyl terminus of the Alzheimer's disease beta A4 amyloid: possible involvement of cathepsin D.

beta A4 amyloid peptide, the main constituent of amyloid plaques and cerebrovascular amyloid deposits associated with Alzheimer's disease, derives from a large precursor protein (APP) by the action of beta- and gamma-secretases, the unidentified endoproteases which release the amino and carboxyl termini of beta A4, respectively. Several gamma-secretase cleavage sites exist which yield the more soluble (1-39/40) forms of beta A4 and the longer forms (1-42/43) which have a greater tendency to aggregate into amyloid plaques. gamma-Secretase activity may therefore be critical in amyloid formation. In this study, a synthetic peptide which encompasses the various gamma-secretase cleavage sites was used as a substrate to probe proteases of various classes and specificities. Elastase, collagenase, and cathepsin D cleaved at the amyloidogenic sites (after Ala42 or after Thr43) to release the carboxyl termini of the aggregating forms. In addition, collagenase and pepsin released the carboxyl terminus of the more soluble forms. Human brain fractions enriched in lysosomes contained a proteolytic activity that cleaved the substrate at the amyloidogenic site(s). This activity was more active at acidic pH and was inhibited by pepstatin, two characteristics of the lysosomal aspartyl proteinase cathepsin D. The same lysosomal fractions were found to contain APP carboxyl-terminal fragments which are potentially amyloidogenic. These were degraded, only in acidic conditions, by an endogenous protease activity inhibited by pepstatin. Thus, a cathepsin D-like activity from human brain is a candidate for APP gamma-secretase(s).

Alzheimer Disease↗
PubMed1995

The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle.

The promastigote surface antigen 2 (PSA-2) complex comprises a family of antigenically similar polypeptides of M(r) 96,000, 80,000 and 50,000, anchored to the membrane with glycosylphosphatidylinositol. Although PSA-2 was initially detected only in promastigotes, Northern blot analysis indicated that mRNA transcripts are also present in amastigotes. Unlike the situation in promastigotes, where at least four major transcripts (2.6-5.3 kb) were detected, only one major (2.6 kb) and two minor transcripts were present in amastigotes. A cDNA clone encoding a member of the PSA-2 family expressed in amastigotes was isolated using DNA probes. The predicted protein sequence of M(r) 40,000 is distinct from promastigote sequences, but shows significant similarity to previously described members of the family from L major and L amazonensis. Antibodies to the carboxyl terminal sequence conserved in all L major PSA-2 studied to date, as well as antibodies affinity purified on the amastigote cDNA-derived polypeptide recognized a major M(r) 50,000 amastigote polypeptide. Immuno-electron microscopy localized both promastigote and amastigote PSA-2 to the cell surface. The expression of PSA-2 polypeptides during the transformation of amastigotes into promastigotes was ordered in a time-dependent manner, with the promastigote M(r) 80000 polypeptide appearing first, followed by the M(r) 96000 polypeptide. In contrast to the glycosylphosphatidylinositol anchor of promastigote PSA-2, which could be hydrolysed by phosphatidylinositol-specific phospholipase C, the amastigote form was resistant to this enzyme.

Amidohydrolases↗
PubMed1994

Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species.

Antibodies raised against a Leishmania major recombinant promastigote surface antigen 2 (PSA-2) fragment recognized three major polypeptides of approximate M(r) 96,000, 80,000 and 50,000 in promastigotes of three Israeli isolates of L. major including the cloned line LRC-L137-V121, but detected a different array of polypeptides in other L. major isolates. The pattern was different both in number of polypeptides detected and their molecular weight. The antibodies to L. major PSA-2 also recognized polypeptides in L. tropica, L. donovani and very weakly in L. mexicana promastigotes and in Crithidia lucilliae. The number and size of the polypeptides was different in each species. In addition to the membrane-bound PSA-2 polypeptides we identified water-soluble forms of PSA-2 released in promastigote culture supernatants. Peptide maps of the various L. major PSA-2 membrane polypeptides showed they were different from each other. N-terminal amino acid sequence of the three polypeptides expressed by L. major showed they are similar but distinct, consistent with being members of a polymorphic family. Because of the extensive sequence similarity between the PSA-2 genes it has been difficult to assign protein products to individual genes. As a first step towards solving this problem, we have transfected into L. mexicana a genomic clone of a L. major PSA-2 gene and shown that it produces a M(r) 35,000 polypeptide recognized by monoclonal and polyclonal antibodies to L. major PSA-2.

Amino Acid Sequence↗
PubMed1994

Ricin-resistant mutants of Leishmania major which express modified lipophosphoglycan remain infective for mice.

Glycosylation variants of the virulent Leishmania major clone V121 were generated by mutagenesis with N-methyl-N-nitroso-N-nitroguanidine and selected using the galactose-specific lectin Ricinus communis II (RCA II). Three mutants, 4B9, 1D1 and 1C12, which failed to bind RCA II, were found to have an altered expression of lipophosphoglycan (LPG), a molecule implicated in the attachment to host macrophages and survival within the phagolysosome. There were differences in the antigenicity, molecular weight and localization of LPG from mutant parasites as compared to V121. Expression of gp63, a surface molecule also implicated in attachment to macrophages, was unaltered. All 3 mutants caused disease when injected into genetically susceptible BALB/c mice but lesions developed at a much slower rate than those caused by the virulent V121 clone. This slow rate of lesion development did not correlate with promastigotes' ability to invade macrophages in vitro. Karyotype analysis showed that there was a reduction in the size of chromosome band number 2 in all 3 mutants. The differences in LPG and chromosome band 2 were retained by mutant clones following passage through mice, suggesting that these phenotypes are stable. Although the mutant parasites were infective and caused lesions, the changed structure of the LPG appeared to influence the virulence of the parasites.

Agglutination↗
PubMed1993

Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major.

This paper describes the cloning of a Golgi-associated GTP-binding protein homologue from Leishmania major. The gene was isolated using degenerate oligonucleotides to conserved sequences amongst the small GTP-binding proteins in a polymerase chain reaction on genomic DNA of the L. major cloned line V121. The reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. A full length copy of the gene was isolated from a lambda gt10 V121 genomic library and sequenced. At the amino acid level the gene showed highest similarity to the human/rat rab1 A gene and the mouse/yeast YPT gene and was named LmYPT. The LmYPT gene was present as a single copy gene in both the L. major and L. donovani genomes. Karyotype analysis localized the LmYPT gene to chromosome band 18 in V121, but it was located on a larger chromosome in the different L. major isolate L119. The LmYPT gene was transcribed as a 3.9-kb transcript in both the promastigote and amastigote forms of the parasite. Western blot analysis, using a polyclonal rabbit antiserum raised against an Escherichia coli expressed portion of the molecule, identified a doublet at 20 and 23 kDa in total promastigote protein. Immunoelectron microscopy in combination with peroxidase staining localized the LmYPT molecule to the Leishmania Golgi apparatus.

Amino Acid Sequence↗
PubMed1992

Cloning and analysis of the RESA-2 gene: a DNA homologue of the ring-infected erythrocyte surface antigen gene of Plasmodium falciparum.

We have cloned and sequenced a homologue of the ring-infected erythrocyte surface antigen (RESA) gene from Plasmodium falciparum designated RESA-2. Two reading frames with high homology to exon 1 and exon 2 of RESA at both the nucleotide and amino acid levels were identified in the RESA-2 sequence. However, RESA-2 does not contain either of the blocks of tandem repeats present in RESA. The lack of an RNA transcript in either asexual or sexual stage parasites and the presence of an in-frame stop codon in the second reading frame suggests RESA-2 could be a pseudogene. Its lack of expression in asexual stages demonstrates that it does not complement the RESA deletion in isolate FCR3.

Amino Acid Sequence↗