B. Frangione

Beta-2 microglobulin is an amyloidogenic protein in man.

Curvilinear fibrils with the tinctorial properties of amyloid were isolated from a patient with bone and joint involvement complicating chronic dialysis for renal disease. Subunit fractions of 24,000 and 12,000 mol wt were identified after gel filtration under dissociating conditions, the latter containing a significant amount of a dimer of the former. This was confirmed by Edman degradation of each fraction, which yielded the amino terminal sequence of normal human beta-2 microglobulin (B2M) to residues 20 and 30, respectively. The size of the subunit protein (12,000 mol wt) and the amino acid composition make it likely that intact B2M is a major constituent of the fibrils. B2M is thus another example of a low molecular weight serum protein, with a prominent beta-pleated sheet structure, that may adopt the fibrillar configuration of amyloid in certain pathologic states.

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.

A soluble monomeric form of Alzheimers 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.

Prion Protein Amyloidosis

The prion protein (PrP) plays an essential role in the pathogenesis of a group of sporadic, genetically determined and infectious fatal degenerative diseases, referred to as “prion diseases”, affecting the central nervous system of humans and other mammals. The cellular PrP is encoded by a single copy gene, highly conserved across mammalian species. In prion diseases, PrP undergoes conformational changes involving a shift from α‐helix to β‐sheet structure. This conversion is important for PrP amyloidogenesis, which occurs to the highest degree in the genetically determined Gerstmann‐Sträussler‐Scheinker disease (GSS) and prion protein cerebral amyloid angiopathy (PrP‐CAA), while it is less frequently seen in other prion diseases. GSS and PrP‐CAA are associated with point mutations of the prion protein gene (PRNP); these conditions show a broad spectrum of clinical presentation, the main signs being ataxia, spastic paraparesis, extrapyramidal signs and dementia. In GSS, parenchyma! amyloid may be associated with spongiform changes or neurofibrillary lesions; in PrP‐CAA, vascular amyloid is associated with neurofibrillary lesions. A major component of the amyloid fibrils in the two diseases is a 7 kDa peptide, spanning residues 81–150 of PrP.

Ten to fourteen residue peptides of Alzheimer's disease protein are sufficient for amyloid fibril formation and its characteristic xray diffraction pattern

The molecular basis of fibril formation in Alzheimers disease was explored by electron micrographic and x-ray diffraction analysis of a series of synthetic peptides corresponding to portions of the amino acid sequence of beta protein and that of its putative precursor. A minimum 14 residue peptide was identified that formed typical amyloid fibrils under physiological conditions. Of these 14 residues, 10 were sufficient to give an identical 4.76 A and 10.6 A diffraction pattern as that recently described for isolated neurofibrillary tangles, amyloid plaque cores and leptomeningeal amyloid fibrils.

Alzheimer's disease and soluble Aβ

Abstract The discovery of soluble amyloid β (sAβ) suggests that the role of amyloid in Alzheimers disease (AD) is similar to the previously studied systemic amyloidoses and alters the notion that membrane damage is the initial event in AD. The disease state is characterized by the abnormal accumulation of a normal degradative peptide, which becomes resistant to further proteolysis due to a conformational change. Mutations in the βPP gene have been found in a very small percentage of AD cases, hence other factors, both genetic and environmental, need to be identified. Priority needs to be given to detailed studies of the structural differences between sAβand the Aβ in amyloid deposits. This will help uncover the determining factors governing the aggregation of sAβ. These structural alterations may be critical for the possible toxic effects Aβ and/or associated proteins (molecular chaperones, e.g., apolipoprotein E) have on brain cell function.

Systemic senile amyloidosis. Identification of a new prealbumin (transthyretin) variant in cardiac tissue: immunologic and biochemical similarity to one form of familial amyloidotic polyneuropathy.

Isolated amyloid fibrils from three cases of systemic senile amyloidosis (SSA) contained subunit proteins with molecular masses of 14 (10-20%), 10-12 (60-80%), and 5-6 kD (5-10%) when fractionated under reducing and dissociating conditions. This grouping was identical to that seen in SKO, a case of familial amyloidotic polyneuropathy (FAP) studied earlier. Amino acid sequencing confirmed that SSA subunit proteins were in fact prealbumin (transthyretin). Complete sequence analysis of one SSA preparation revealed the presence of a new variant Pa (TTr) molecule with a single amino acid substitution of isoleucine for valine at position 122. Further studies used an antiserum specific for SKO IV, a subunit protein of SKO previously shown to correspond to carboxy-terminal 78 residues (positions 49-127) of (TTr). Anti-SKO IV reacted with SSA in tissue at equivalent dilutions to anti-Pa (TTr) and with the 10-12-kD fraction of SSA on Western blots; reactivity was blocked by SKO IV, but not by Pa (TTr). SSA is a form of systemic amyloidosis caused by tissue deposition of Pa (TTr) and its fragments, with shared conformational or subunit antigenicity to at least one form of FAP. Identification of a new variant Pa (TTr) molecule in one case suggests further that SSA may be a genetically determined disease expressed late in life.

Substitution at codon 22 reduces clearance of Alzheimer’s amyloid-β peptide from the cerebrospinal fluid and prevents its transport from the central nervous system into blood

A point mutation of G to C at codon 693 of the amyloid-beta (Abeta) precursor protein gene results in Glu to Gln substitution at position 22 of the Abeta (AbetaQ22) associated with hereditary cerebrovascular amyloidosis with hemorrhage Dutch type. Factors that regulate AbetaQ22 levels in the central nervous system (CNS) are largely unknown. By using ventriculo-cisternal perfusion technique in guinea pigs, we demonstrated that clearance from the cerebrospinal fluid and transport from the CNS to blood of [(125)I]-AbetaQ22 (1 nM) were reduced by 36% and 52%, respectively, in comparison to the wild type Abeta(1-40) peptide. In contrast to significant uptake and transport of Abeta(1-40) across the brain capillaries and leptomeningeal vessels, AbetaQ22 was not taken up at these CNS vascular transport sites, which was associated with its 53% greater accumulation in the brain. The CNS clearance of Abeta(1-40) was half-saturated at 23.6 nM; AbetaQ22 had about 6.8-fold less affinity for the CNS efflux transporters and its elimination relied mainly on transport across the choroid plexus. Thus, the Dutch mutation impairs elimination of Abeta from brain by reducing its rapid transport across the blood-brain barrier and the vascular drainage pathways, which in turn may result in accumulation of the peptide around the blood vessels and in brain.

STUDIES ON ALPHA CHAIN DISEASE

Since the initial descripition of alpha chain disease (a-C.D.) in 1968,l this disorder has been recognized in 21 patients to our knowledge. Most cases have been detected or authenticated in our laboratory. Although the first case of heavy y chain disease was reported in 1964,? the number of recorded patients with this condition is smaller.3 Thus a! chain disease is probably the most frequent disorder among the group of heavy chain diseases (H.C.D.) which, at present, includes diseases of the y, a and p 4 types. The clinicopathological features were strikingly similar in all recorded patients with a chain disease except one. As in the first patient,5 the lymphoplasmacytic proliferation involved primarily the whole length of the small intestine and was associated with severe malabsorption.

Intact Alzheimer amyloid precursor protein (APP) is present in platelet membranes and is encoded by platelet mRNA

Using antibodies directed against N-terminal and C-terminal epitopes we have immunologically detected APP species in the membrane and saline-soluble fractions of unstimulated platelets, and in the conditioned medium of thrombin-stimulated platelets. These studies demonstrate an intact 140 kD membrane-associated form of APP that is released on degranulation. Evidence that platelets synthesize at least one form of APP (APP751) was obtained by enzymatic amplification of specific mRNA using Polymerase Chain Reaction (PCR) and direct sequence analysis of PCR product. Processing of APP for release may occur via successive C-terminal truncations, and/or by the release and proteolysis of an intact membrane associated form. An intact form of APP in platelets provides a circulating substrate upon which proteases from many tissues may act to produce beta protein (AB) during pathologic conditions.

Staphylococcal Protein A and Human IgG Subclasses and Allotypes

Staphylocoecal protein A binds molecules belonging to the IgGl, IgG2, and IgG4 sub‐classes. IgG3 proteins generally do not bind, except for those coded by the two γ3 alleles, which are G3m(u−): G3m(b0,b3,b5,s,v) and G3m(b0,b3,b5,s,t,v). G3m(u) is located in the CH2 domain. The difference between G3m(u−) and G3m(u+) IgG3 proteins correlates with the sequence at position 339 in the CH2 domain—Ala and Thr respectively. There is another structural difference in the CH3 domain which correlates with protein A binding and non‐binding: all IgG proteins that bind protein A have His at position 435, whereas those that do not, have Arg at that position.