Search PubMed⌕ Search

Biomedical subjects

C G Glabe

Publications and source records attributed to C G Glabe.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cell surface APP751 forms complexes with protease nexin 2 ligands and is internalized via the low density lipoprotein receptor-related protein (LRP).

The secreted isoforms of the amyloid precursor protein (APP) that contain the Kunitz domain are also known as protease nexin 2 (PN2). Normal proteolytic processing of transmembrane APP, which results in the majority of soluble PN2, cleaves within the Alzheimer's A beta peptide, precluding A beta formation. Recent data indicate that soluble PN2 is internalized by cells via the low density lipoprotein receptor-related protein (LRP), which binds multiple ligands including apolipoprotein E (apoE) [23]. However, soluble PN2 cannot contribute to amyloid accumulation, so we examined whether the unprocessed, transmembrane form of APP751 containing the intact A beta sequence would form complexes with a PN2 ligand, EGF binding protein (EGFBP), and be internalized by LRP. We found that the addition of EGFBP to cells overexpressing APP751 induced the internalization of this amyloidogenic form of APP. The 39 kDa LRP receptor associated protein (RAP), an antagonist for LRP, blocked the internalization of APP751/PN2, suggesting a common LRP-mediated internalization pathway for both soluble and transmembrane APP751/PN2 after protease complex formation. Previous work has shown that internalization of transmembrane APP can lead to the formation of amyloidogenic carboxyl-terminal fragments and increased secretion of the Alzheimer's A beta peptide. Our data suggest the protease ligands for PN2 may play an important role in altering APP processing pathways to favor amyloid formation, and that LRP may be a point at which the apoE and amyloid processing pathways intersect.

Amyloid beta-Protein Precursor↗
PubMed1996

The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein, PKD1.

During fertilization, the sea urchin sperm acrosome reaction (AR), an ion channel-regulated event, is triggered by glycoproteins in egg jelly (EJ). A 210-kD sperm membrane glycoprotein is the receptor for EJ (REJ). This conclusion is based on the following data: purified REJ binds species specifically to EJ dotted onto nitrocellulose, an mAb to REJ induces the sperm AR, antibody induction is blocked by purified REJ, and purified REJ absorbs the AR-inducing activity of EJ. Overlapping fragments of REJ cDNA were cloned (total length, 5,596 bp). The sequence was confirmed by microsequencing six peptides of mature REJ and by Western blotting with antibody to a synthetic peptide designed from the sequence. Complete deglycosylation of REJ followed by Western blotting yielded a size estimate in agreement with that of the mature amino acid sequence. REJ is modular in design; it contains one EGF module and two C-type lectin carbohydrate-recognition modules. Most importantly, it contains a novel module, herein named the REJ module (700 residues), which shares extensive homology with the human polycystic kidney disease protein (PKD1). Mutations in PKD1 cause autosomal dominant polycystic kidney disease, one of the most frequent genetic disease of humans. The lesion in cellular physiology resulting from mutations in the PKD1 protein remains unknown. The homology between REJ modules of the sea urchin REJ and human PKD1 suggests that PKD1 could be involved in ionic regulation.

Amino Acid Sequence↗
PubMed1995

A sparse matrix screen to establish initial conditions for protein renaturation.

Protein renaturation is of particular interest not only for the basic mechanisms of protein folding but also as a practical problem for proteins overexpressed in microorganisms, since recombinant proteins may accumulate as misfolded aggregates in "inclusion bodies" that are inactive after purification. We have established a systematic screening method to identify conditions which promote protein renaturation. A matrix of 50 different buffers, which were originally developed for protein crystallization, were found to facilitate the renaturation for eight of nine different proteins examined. The proteins tested include the adhesive protein bindin, recombinant bindin, and a variety of enzymes, including bacterial alkaline phophatase, horseradish peroxidase, lysozyme, trypsin, beta-galactosidase, rabbit carboxylesterase, and acetylcholinesterase. The total amount of activity recovered varied from 9 to 333% depending on the protein. The conditions that were found to promote renaturation are very different from the optimal conditions for enzyme activity. The finding that most of the proteins tested renatured to a significant extent in one or more of the buffers in the matrix suggests that the sparse matrix screen may be of general utility for establishing initial renaturation conditions for a wide variety of proteins. One initial renaturation conditions have been identified, the conditions may be optimized by systematically altering other parameters of the renaturation process.

Acetylcholinesterase↗
PubMed1995

Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25-35 region to aggregation and neurotoxicity.

The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of beta-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of beta-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of beta-amyloid, beta 25-35, and the full-length protein, beta 1-42. We examine the effects of amino acid residue deletions and substitutions on the ability of beta-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant beta-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of beta 25-35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the beta 33-35 region. In beta 1-42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of beta-sheet secondary structure in aggregating, toxic beta-amyloid peptides but not in nonaggregating, nontoxic beta-amyloid peptides. Together, these data further define the primary and secondary structures of beta-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.

Alzheimer Disease↗
PubMed1994

beta-Amyloid activates complement by binding to a specific region of the collagen-like domain of the C1q A chain.

beta-amyloid peptides that accumulate within the brain of individuals with Alzheimer's disease bind to C1q and activate the classical C pathway via a specific interaction with a site within the collagen-like domain of C1q (C1q-CLF). Synthetic analogues of beta-amyloid peptides, beta 1-42 and beta 1-40, bound to C1q and were strong activators of C as assessed by both total C consumption and C4 consumption. beta 1-42 was significantly more effective than beta 1-40 in binding to C1q and triggering C activation, whereas beta 1-28 demonstrated little or no binding or C activation. This C-activating capacity seems to be largely correlated with the assembly of the beta 1-42 into low speed sedimentable aggregates and/or macromolecular fibrils. Radiolabeled C1q and C1q-CLF bind specifically to these aggregates or amyloid fibrils. In addition, using synthetic C1q peptides in a solid phase binding assay, the major binding site of beta 1-42 to C1q was localized to the C1q A chain collagen-like residues 14-26, a region previously described as a novel interaction site for Ab-independent activators of C1. C1q A chain peptide 14-26 blocked the ability of beta-amyloid peptides to activate the classical C pathway, providing evidence that this relatively unrecognized mechanism of C activation (via binding to the C1q-CLF) may have crucial physiologic consequences. Finally, these observations provide further support for the hypothesis that C activation and inflammation may be a component in the pathogenesis of AD and suggest possibilities for modulating the progression of AD.

Alzheimer Disease↗
PubMed1993

Characterization of the membrane-associating domain of the sperm adhesive protein, bindin.

Bindin is an adhesive protein that mediates the binding of sea urchin sperm to the egg during fertilization. Bindin selectively associates with gel-phase phospholipid vesicles in a peripheral fashion. Bindin interacts specifically with sulfated fucan on the egg's surface, and directly with the phospholipid bilayer of the sperm. Analysis of a series of deletion mutants of recombinant bindin was undertaken to define the membrane associating domain of bindin. Recombinant and sperm bindin display nearly identical binding kinetics to gel-phase phospholipids and have equivalent saturation points of approx. 250 lipid molecules per molecule of bindin. Deletion mutants of bindin which contain residues 75-130 retained specific membrane binding activity. Synthetic peptides corresponding to residues 69-130, and 92-130 also display gel-phase specific membrane association. This region is highly conserved within four different species of bindin molecules. Circular dichroism spectroscopy of synthetic peptides corresponding to residues 92-130 and 69-130 suggests that a distinct change in conformation takes place upon binding liposomes. Taken together, these data indicate that the membrane binding activity of bindin residues within this highly conserved region of the bindin molecule.

Amino Acid Sequence↗
PubMed1993

Structure/function analysis of the sea urchin sperm adhesive protein bindin.

The functional domain structure of bindin, an adhesive protein component of the acrosomal granule of sea urchin sperm, has been analyzed using a series of carboxyl- and amino-terminal deletion analogs of recombinant bindin expressed in Escherichia coli. Bindin, which self-associates to form macromolecular aggregates, mediates the species-specific attachment of sperm to the egg surface as well as egg-to-egg agglutination in vitro by binding to egg surface-sulfated fucose-containing glycoconjugates. Cross-species egg agglutination activity and sperm adhesion were determined for two sea urchin species, Strongylocentrotus purpuratus and S. franciscanus. We found that S. franciscanus sperm bindin agglutinates both S. franciscanus and S. purpuratus eggs whereas S. purpuratus sperm bindin displays a much more restricted specificity for homologous S. purpuratus eggs. Consistent with this result, cross-species sperm adhesion experiments demonstrate that S. franciscanus sperm also bind more efficiently to S. purpuratus eggs in comparison to S. purpuratus sperm attachment to S. franciscanus eggs. Recombinant S. purpuratus bindin demonstrates the same species-specific egg agglutination properties observed for sperm bindin. Deletion mutants of S. purpuratus bindin were tested for their ability to species specifically agglutinate eggs. We found that residues 1-74 or residues 122-236 can be deleted without a loss of the species-specific agglutination properties of bindin. Comparison of the bindin amino acid sequences from the two species reveals that all of the active deletion analogs have a central segment in common corresponding to residues 75 through 121 of S. purpuratus bindin, where the sequences are identical except for a single amino acid, Arg 77, which is an Ala residue in S. franciscanus. However, substitution of Arg 77 for Ala does not alter the species specificity of recombinant bindin, suggesting that the more divergent amino- and carboxy-terminal sequences flanking the conserved central domain determine the species specificity of bindin and either end is sufficient to impart specificity. The amino- and carboxyl-terminal thirds of S. purpuratus bindin each contain two repeats of the sequence M G G P P/V. This sequence does not occur in S. franciscanus, which instead has five repeats of the sequence M G G A/V/Q F/V/P. It is conceivable that these repeats, which vary in sequence in bindins from different species, constitute an important part of the species recognition mechanism.

Agglutination↗
PubMed1993

In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor.

Recent genetic experiments have suggested that tat transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat requires functional upstream enhancer sequences--Sp1 sites, in particular. In these experiments, HeLa cell nuclear extracts were passed over affinity matrices containing chemically synthesized or bacterially expressed HIV-1 Tat. Assay of material that bound to and eluted from the Tat matrices revealed the presence of the Sp1 transcription factor. Other transcription factors (Oct and NF-kappa B) also bound to Tat matrices but with less efficiency--in parallel with the lower capacities of these binding motifs to confer Tat responsiveness on a basal HIV-1 promoter compared with Sp1 sites. Passage of nuclear extracts over matrices containing other neutral proteins, including bovine serum albumin, ovalbumin, and lysozyme, revealed no or reduced binding. Cross-linking experiments indicated that the purified Sp1 and Tat proteins can form multimeric complexes in the absence of other proteins. The region of Tat responsible for Sp1 binding was localized to a region encompassing residues 30 to 62. Immunoprecipitation experiments with HIV-1-infected T lymphocytes indicated coimmunoprecipitation of Tat and Sp1. These experiments extend previous genetic experiments and suggest a direct interaction between Tat and Sp1 during transactivation.

Base Sequence↗
PubMed1993

Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state.

The progressive neurodegeneration of Alzheimer's disease has been hypothesized to be mediated, at least in part, by beta-amyloid protein. A relationship between the aggregation state of beta-amyloid protein and its ability to promote degeneration in vitro has been previously suggested. To evaluate this hypothesis and to define a structure-activity relationship for beta-amyloid, aggregation properties of an overlapping series of synthetic beta-amyloid peptides (beta APs) were investigated and compared with beta AP neurotoxic properties in vitro. Using light microscopy, electrophoresis, and ultracentrifugation assays, we found that few beta APs assembled into aggregates immediately after solubilization, but that over time peptides containing the highly hydrophobic beta 29-35 region formed stable aggregations. In short-term neuronal cultures, toxicity was associated specifically with those beta APs that also exhibited significant aggregation. Further, upon the partial reversal of beta 1-42 aggregation, a concomitant loss of toxicity was observed. A synthetic peptide derived from a different amyloidogenic protein, islet amyloid polypeptide, exhibited aggregation but not toxicity, suggesting that beta AP-induced neurotoxicity in vitro is not a nonspecific reaction to aggregated protein. The correlation between beta AP aggregation and neurotoxicity was also observed in long-term neuronal cultures but not in astrocyte cultures. These data are consistent with the hypothesis that beta-amyloid protein contributes to neurodegeneration in Alzheimer's disease.

Amino Acid Sequence↗
PubMed1992

Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/beta protein.

The A4 or beta protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/beta protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, beta 1-42, corresponding to one of the longer forms of the A4/beta protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the beta 1-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular beta 1-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/beta protein may be resistant to degradation and suggest that the A4/beta protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/beta protein if this part of APP is selectively resistant to proteolysis.

Amino Acid Sequence↗
PubMed1991

In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity.

beta-Amyloid peptide forms the senile plaques of Alzheimer's disease and has been previously demonstrated to have both trophic and toxic effects on neurons in vitro. We report here that synthetic beta-amyloid peptide shows both aggregation and neurotoxicity after a 2-4 day incubation period, but is neurite-promoting and not toxic in its initially solubilized state. SDS-PAGE characterization shows that newly solubilized beta-amyloid is predominantly monomeric whereas incubated peptide has several high molecular weight species.

Amyloid beta-Peptides↗
PubMed1991

Site-specific incorporation of nonnatural residues during in vitro protein biosynthesis with semisynthetic aminoacyl-tRNAs.

A method is presented for the incorporation of nonnatural amino acids into proteins during in vitro cell-free translation. A combination of chemical synthesis and run-off transcription was employed to prepare a semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressor acylated with L-3-[125I]iodotyrosine. The presence of this synthetic tRNA during in vitro translation of mRNA containing a nonsense suppression site (e.g., a UAG termination codon) results in the incorporation of the nonnatural amino acid L-3-iodotyrosine into the polypeptide exclusively at the position corresponding to that site. Incorporation of the nonnatural amino acid L-3-[125I]iodotyrosine into the model polypeptide was assessed by quantitative and unambiguous determination of suppression efficiency, read-through, and site specificity of incorporation. Minor modifications of the method employed in this initial experiment also allow the rapid analysis of unlabeled acylated tRNA analogues. Under optimum conditions, the unlabeled amino acid L-3-iodotyrosine was found to be incorporated with a suppression efficiency of 65%. Other nonnatural residues, including N-methylphenylalanine, D-phenylalanine, and phenyllactic acid, were tested in the assay under these same conditions. Suppression efficiencies for this series ranged from 0 to 72% depending on the structure of the residue incorporated. Several other aspects of this methodology, such as tRNA structure and context effects, are briefly discussed.

Acylation↗
PubMed1991

The sequence of the Arbacia punctulata bindin cDNA and implications for the structural basis of species-specific sperm adhesion and fertilization.

Bindin is the major protein component of the acrosome granule of sea urchin sperm which mediates the species-specific adhesion of sperm to the egg surface during fertilization. Bindin isolated from both Arbacia punctulata and Strongylocentrotus purpuratus sperm demonstrate a distinct adhesive preference for eggs of the same species although a significant amount of cross-species reactivity is observed. Here we describe the isolation and sequence of A. punctulata bindin cDNA clones and a comparison of the predicted protein sequence with the sequence previously reported for S. purpuratus bindin (Gao et al., 1986, Proc. Natl. Acad. Sci., USA 83, 8634-8638). Bindins from these genera show substantial sequence similarity in both the mature bindin domain and the probindin precursor region. The most striking identity is a region of 42 conserved amino acids in the central part of the mature bindins. This conserved domain may be responsible for conserved functions of bindin. Regions flanking this conserved element on both the amino and carboxyl side are more highly divergent, suggesting that they are responsible for the species-specific properties of bindin. The mature A. punctulata sequence contains a putative transmembrane segment between residues 431 and 451 that is absent from S. purpuratus bindin. This structural element may account for the previous observation that isolated A. punctulata bindin uniquely forms multilamellar structures reminiscent of lipid bilayers and binds significant amounts of phospholipid and detergent. The structure of this hydrophobic segment also displays a number of similarities to viral fusion peptides.

Amino Acid Sequence↗
PubMed1990

Beta-amyloid protein promotes neuritic branching in hippocampal cultures.

In the neuritic plaques of Alzheimer's disease, abnormal neuritic processes cluster around a core of beta-amyloid protein. Previous data have shown that beta 1-28, a peptide homologous to the first 28 amino acid residues of beta-amyloid protein, enhanced survival without affecting neuritic extension or branching in cultures of hippocampal neurons. In this paper we show that beta 1-42, a synthetic peptide which corresponds to the full 42 amino acid sequence of beta-amyloid protein, increased cell survival and also promoted the elongation of axon-like processes, raised the number of dendrite-like processes, and increased their arborization.

Amyloid↗
PubMed1990

Specific recognition of sulfate esters by bindin, a sperm adhesion protein from sea urchins.

Bindin specifically binds to egg surface sulfated fucan polysaccharides and mediates the attachment of sperm to the egg during fertilization. Sulfate esters are critical for this interaction. We have examined the effect of different anionic groups on the relative binding affinities of a series of homologous anionic polymers for bindin to determine the extent to which other charged moieties can substitute for sulfate. We found that bindin displays a remarkable specificity for sulfate- or sulfonic acid-containing polymers. The relative affinities of poly(vinyl sulfate) and poly(styrenesulfonic acid) are four orders of magnitude higher than polymers containing phosphate esters or carboxyl groups. The bindin-mediated aggregation of sea urchin eggs was inhibited by the sulfated polymers but not the other anionic polymers. This high degree of selectivity for sulfated polymers is not observed for the binding of the polyanions to most other proteins and basic polypeptides. These results suggest that the binding is not due to the formation of simple salt bridges, and that all three non-ester oxygen atoms of the sulfate groups are involved in multiple bonding interactions with a complementary 'docking site' on the bindin polypeptide. The orientation of the polysaccharide sulfate oxygen atoms relative to the protein binding site may be an important determinant of the specificity of polysaccharide binding.

Animals↗
PubMed1990

Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1.

Full-length (86-residue) polypeptide corresponding to the human immunodeficiency virus type 1 tat trans-activating protein was chemically synthesized on a semiautomated apparatus, using an Fmoc amino acid continuous-flow strategy. The bulk material was relatively homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, and it showed trans-activating activity when scrape loaded into cells containing a human immunodeficiency virus long terminal repeat-chloramphenicol acetyl-transferase reporter plasmid. Reverse-phase high-pressure liquid chromatography yielded a rather broad elution profile, and assays across the column for biological activity indicated a sharper peak. Thus, high-pressure liquid chromatography provided for enrichment of biological activity. Fast atom bombardment-mass spectrometry of tryptic digests of synthetic tat identified several of the predicted tryptic peptides, consistent with accurate chemical synthesis.

Amino Acid Sequence↗
PubMed1989

Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin.

We have investigated the domain of the bindin polypeptide that selectively associates with gel-phase phospholipid vesicles. We found that small trypsin fragments of bindin retain the ability to selectively associate with gel-phase vesicles. The primary amino acid sequence of bindin suggests that these peptides are derived from the central portion of the polypeptide between residues 77 and 126, which is the most hydrophobic region of bindin. We have also employed 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and novel, radioiodinated, photoactivatable derivatives of the polar head group of phosphatidylethanolamine (ASD-PE and ASA-PE) to identify membrane-associated polypeptide segments after the transfer of radiolabel from the probe to the bindin polypeptide. After photolysis, bindin was selectively labeled only from probes incorporated in gel-phase vesicles. The labeling of bindin was much more efficient from the head group probes ASA-PE and ASD-PE (8 and 2% of the total label, respectively) in comparison to the hydrophobic probe TID (less than 0.02% of the total label), suggesting that bindin is localized within the polar part of the bilayer. Protease mapping experiments with V8 protease, trypsin, and endoprotease Lys-C suggest that some of the probe label is distributed along the amino-terminal portion of bindin between residues 1 and 76 and the rest of the label is restricted to the segments between residues 77 and 126 which also selectively bind to gel-phase vesicles. The carboxyl-terminal portion of bindin between residues 127 and 236 is not labeled.

Amino Acid Sequence↗