Rieko Oyama

Morquio disease: Isolation, characterization and expression of full-length cDNA for human N-acetylgalactosamine-6-sulfate sulfatase

We cloned and sequenced a full-length cDNA of human placental N-acetylgalactosamine-6-sulfate sulfatase, the enzyme deficient in Morquio disease. The 2339-nucleotide sequence contained 1566 nucleotides which encoded a polypeptide of 522 amino acid residues. The deduced amino acid sequence was composed of a 26-amino acid N-terminal signal peptide and a mature polypeptide of 496 amino acid residues including two potential asparagine-linked glycosylation sites. Expression of the cDNA in transfected deficient fibroblasts resulted in higher production of this sulfatase activity than in untransfected deficient fibroblasts. The cDNA clone was hybridized to only a 2.3-kilobase species of RNA in human fibroblasts. The amino acid sequence of N-acetylgalactosamine-6-sulfate sulfatase showed a high degree of homology with those of other sulfatases such as human arylsulfatases A, B or C, glucosamine-6-sulfatase, iduronate-2-sulfatase and sea urchin arylsulfatase.

Down's Syndrome: Up-Regulation of β-Amyloid Protein Precursor and τ mRNAs and Their Defective Coordination

Abstract: Almost all patients >40 years of age with Downs syndrome (DS) develop the pathology characteristic of Alzheimers disease: abundant β‐amyloid plaques and neurofibrillary tangles. We have investigated the gene expression of β‐amyloid protein precursor (APR) and τ in DS and age‐matched control brains and found that levels of both mRNAs were significantly elevated in DS. Such up‐regulation was not observed in two other neuronal proteins. A correlation between total APP and τ mRNA levels was also found in DS brain but distinct from the pattern observed in normal brain. Although a proportionality existed between APP‐695 mRNA and three‐repeat τ mRNA in DS, the proportionality between APP‐751 mRNA and four‐repeat τ mRNA, which is normally present, was not observed. Thus, DS brains are primarily characterized by the up‐regulation of τ mRNA as well as APP mRNA and disruption of the coordinate expression between APP‐751 and four‐repeat τ.

Glutamine synthetase, hemoglobin α-chain, and macrophage migration inhibitory factor binding to amyloid β-protein: their identification in rat brain by a novel affinity chromatography and in Alzheimer’s disease brain by immunoprecipitation

Proteins binding to amyloid beta-protein (Abeta) may modulate the accumulation of Abeta in Alzheimers disease (AD) brain. We developed a monomeric Abeta column for isolation of the proteins binding to Abeta from rat brain. By amino acid sequence analysis and immunoreactivity with specific antibodies, we identified three new Abeta-binding proteins, glutamine synthetase, hemoglobin alpha-chain, and macrophage migration inhibitory factor as well as serum albumin, beta-tubulin, and glyceraldehyde-3-phosphate dehydrogenase already identified as proteins bound to amyloid beta-protein precursor. In addition, the retained fraction contained both apolipoprotein E and alpha(1)-antichymotrypsin already known as Abeta binding proteins. Furthermore, we detected the complexes of these new binding proteins with Abeta in a soluble fraction of the cerebral cortex of AD brain by immunoprecipitation. Our results suggest that these binding proteins also associate with Abeta, leading to the clearance or the accumulation of Abeta and the neuronal cell damage in human brain.

Purification and complete amino acid sequence of novel β2-microglobulin

Abstract We have previously reported that novel β2-microglobulin (β2m) is a metabolite derived from β2m in ultrafiltrate of patients on long-term hemodialysis (LT-HD) [1]. Chromatofocusing showed the presence of at least two major novel β2ms with isoelectric points of 5.38 and 5.22. In the present study we purified one of major novel β2ms and determined the complete amino acid sequence. We demonstrate herein that the novel β2m has the same sequence as native β2m except for the 17th residue from the N-terminus which was identified as Asp instead of Asn in native β2m, suggesting a possible deamidation during LT-HD.

Apolipoprotein E genotype, Alzheimer's pathologies and related gene expression in the aged population

We have investigated the effect of genotypes of apolipoprotein E (ApoE) on the pathologies found in Alzheimers disease (AD) and its related gene expression in 38 aged human brains obtained from consecutive autopsied cases. ApoE2/3, -3/3, -3/4, and -4/4 were typed in those aged brains, with ApoE3/3 being most prevalent. The AD pathologies were undetectable in ApoE2/3 brains, but were frequently observed in the other ApoE groups. In ApoE3/3 brains, 55%, 34%, and 24% of the cortical sections examined showed senile plaques (SPs), neurofibrillary tangles (NFTs), and cerebral amyloid angiopathy (CAA), respectively. In ApoE4/4 brains, the SP formation was significantly higher. The ApoE genotype neither affected ApoE, APP, or tau mRNA level, nor the differential expression of the latter two. These results suggest that ApoE4/4 accelerates and ApoE2/3 decelerates the development of the AD pathologies in the aged brain, but this is not through alterations of the APP and tau gene expression.

Differential expression of β amyloid protein precursor (APP) and tau mRNA in the aged human brain : individual variability and correlation between APP-751 and four-repeat tau

We investigated the relationship between the differential expression of β amyloid protein precursor (APP) and tau mRNA, and the extent of β and tau deposition in three regions from each of the 38 aged brains obtained from consecutive autopsied cases. Remarkable variabilities were noted in the ratios of APP-770/-751/ −695 and four-repeat tau among elderly individuals. There was no consistent alteration in the APP differential expression among β plaque (-), (+), and (+ +-+ + +) groups. Also, no differences in the four-repeat tau ratios were noted among tangle (-), (+), and (++) groups. Despite these great individual variabilities, APP-751 was found to be well-correlated with four-repeat tau. It is possible that APP-751 and four-repeat tau are increasing during aging, while APP-695 and three-repeat tau are decreasing.

β‐Amyloid Protein Precursor and τ mRNA Levels Versus β‐Amyloid Plaque and Neurofibrillary Tangles in the Aged Human Brain

Abstract: To learn whether or not the levels of β‐amyloid protein precursor (APP) and τ mRNAs are related to the formation of β‐amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocyto‐chemically for β‐amyloid and tangles. Marked individual variabilities were noted in APP and τ mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the β‐amyloid plaque (++) group, but not in the plaque (+) group, compared to the plaque (−) group. Some brains in the plaque (−) group showed increased APP expression, the extent of which was not seen in the plaque (+)or(++) group. The differences in the mean τ mRNA levels were not statistically significant among the tangle (−), (+), and (++) groups. These results show that β‐protein and τ deposition do not accompany increased expression of the APP and τ genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of β‐amyloid and/or tangle formation in the aged human brain.

The complete amino acid sequence of the mature form of rat sepiapterin reductase

The partial amino acid sequence of rat sepiapterin reductase was determined using peptides generated by cleavage of the S-carboxyamidomethylated protein with Achromobacter protease I, cyanogen bromide, chymotrypsin or BNPS-skatole. The protein began with N-acetyl methionyl residue at the N-terminus and ended with isoleucyl residue at the C-terminus. The present results essentially coincided with the amino acid sequence predicted from the nucleotide sequence of the cDNA recently reported by Citron et al. (Proc. Natl. Acad. Sci. USA 87, 6436-6440 (1990)), clarified the processing event during the biosynthesis and provided the complete amino acid sequence of the mature form of the enzyme.

A Novel Correlation Between the Levels of β‐Amyloid Protein Precursor and τ Transcripts in the Aged Human Brain

Abstract: β‐Amyloid protein precursor (APP) and τ are implicated in the pathogenesis of Alzheimers disease. We quantified the levels of APP and τ transcripts in the three cortical regions of 38 aged human brains obtained from consecutive autopsied patients. The level of APP mRNA was directly proportional to that of τ mRNA to a remarkable extent, suggesting coordinate expression of the APP and τ genes, whereas much weaker correlations were noted among mRNAs encoding other neuronal proteins. From the previous data on the differential expression of APP and τ mRNAs, the levels of APP‐751 and ‐695 mRNAs were calculated and found to be proportional to those of four‐repeat and three‐repeat τ mRNAs, respectively, whereas that of APP‐770 mRNA was rather constant. These results suggest that the mRNA concentrations of APP isoforms are linked to those of τ isoforms in the aged human brain.