γ Heavy Chain Disease in Man: cDNA Sequence Supports Partial Gene Deletion Model

Human gamma heavy chain disease (HCD) is characterized by the presence in serum of a short monoclonal Ig gamma chain unattached to light chains. Although most HCD proteins have internal deletions, in some the defect is NH2-terminal. The OMM gamma 3 HCD serum protein is of the latter type, having undergone an extensive NH2-terminal deletion with a sequence starting within the hinge. A cell line synthesizing the OMM protein has enabled us to study the biogenesis of the abnormal molecule. In vitro translation of isolated mRNA yields a protein containing a hydrophobic NH2-terminal leader sequence. In the intact cell, the precursor molecule is processed normally to yield a protein with an NH2-terminal sequence homologous to the beginning of the variable (V) region. The nucleotide sequence of cDNA prepared from the OMM mRNA encodes a 19-amino acid leader followed by the first 15 residues of the V region. An extensive internal deletion encompasses the remainder of the V and the entire CHl domain. Immediately following the short V region, there is information in the cDNA for the entire normal hinge. The primary synthetic product is thus an internally deleted molecule that undergoes postsynthetic degradation to yield the NH2-terminally deleted serum protein. The structure of the OMM mRNA suggests that the protein abnormality results from a partial gene deletion rather than defective splicing

Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.Fil: Morelli, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Llovera, Ramiro Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez, Silvia Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Affranchino, Jose Luis. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Prelli, Frances. University of New York; Estados UnidosFil: Frangione, Blas. University of New York; Estados UnidosFil: Ghiso, Jorge. University of New York; Estados UnidosFil: Castaño, Eduardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Identification of distinct N-terminal truncated forms of prion protein in different Creutzfeldt-Jakob disease subtypes.

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules

Human blood-brain barrier receptors for Alzheimer's amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer.

This is the published version. Copyright 1998 by American Society for Clinical Investigation.A soluble monomeric form of Alzheimer's amyloid-beta (1-40) peptide (sAbeta1-40) is present in the circulation and could contribute to neurotoxicity if it crosses the brain capillary endothelium, which comprises the blood-brain barrier (BBB) in vivo. This study characterizes endothelial binding and transcytosis of a synthetic peptide homologous to human sAbeta1-40 using an in vitro model of human BBB. 125I-sAbeta1-40 binding to the brain microvascular endothelial cell monolayer was time dependent, polarized to the apical side, and saturable with high- and low-affinity dissociation constants of 7.8+/-1.2 and 52.8+/-6.2 nM, respectively. Binding of 125I-sAbeta1-40 was inhibited by anti-RAGE (receptor for advanced glycation end products) antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage scavenger receptor (SR), type A, displayed binding and internalization of 125I-sAbeta1-40. The internalized peptide remains intact > 94%. Transcytosis of 125I-sAbeta1-40 was time and temperature dependent, asymmetrical from the apical to basolateral side, saturable with a Michaelis constant of 45+/-9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. We conclude that RAGE and SR mediate binding of sAbeta1-40 at the apical side of human BBB, and that RAGE is also involved in sAbeta1-40 transcytosis

Copper chelation delays the onset of prion disease.

The prion protein (PrP) binds copper and under some conditions copper can facilitate its folding into a more protease resistant form. Hence, copper levels may influence the infectivity of the scrapie form of prion protein (PrP Sc ). To determine the feasibility of copper-targeted therapy for prion disease, we treated mice with a copper chelator, D-(؊)-penicillamine (D-PEN), starting immediately following intraperitoneal scrapie inoculation. D-PEN delayed the onset of prion disease in the mice by about 11 days (p ‫؍‬ 0.002), and reduced copper levels in brain by 29% (p < 0.01) and in blood by 22% (p ‫؍‬ 0.03) compared with control animals. Levels of other metals were not significantly altered in the blood or brain. Modest correlation was observed between incubation period and levels of copper in brain (p ‫؍‬ 0.08) or blood (p ‫؍‬ 0.04), indicating that copper levels are only one of many factors that influence the rate of progression of prion disease. In vitro, copper dose-dependently enhanced the proteinase K resistance of the prion protein, and this effect was counteracted in a dose-dependent manner by co-incubation with D-PEN. Overall, these findings indicate that copper levels can influence the conformational state of PrP, thereby enhancing its infectivity, and this effect can be attenuated by chelatorbased therapy