Biomedical subjects
Publications and source records attributed to C Fukazawa.
We created three lines of transgenic mice with an integrated rat genomic apolipoprotein E gene fused with the mouse metallothionein I promoter. These lines transcribed rat apoE mRNA in the liver and/or in the kidney and expressed significant amounts of rat apoE in plasma. Enhancement of the plasma level by treatment with Zn ion or Bi ion was observed.
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To study the organ-specific effect of probucol, a potent cholesterol-lowering drug, on apolipoprotein(apo)E synthesis, rabbit apoE complementary deoxyribonucleic acid (cDNA) clones were isolated and apoE messenger ribonucleic acid (mRNA) levels were determined in various tissues isolated from probucol-treated rabbits. A 0.54 kb apoE cDNA was cloned from a rabbit liver cDNA library using a synthetic oligonucleotide probe. The amino acid sequence deduced from an apoE coding region indicated 78% homology for human apoE and 63% homology for rat apoE. RNA blot analysis showed that apoE mRNA was most abundant in the liver, then in the brain and spleen. The relative amounts of apoE mRNA were determined in tissues of rabbits fed a normal diet, or high-cholesterol (1%) diet with or without probucol (1%). ApoE mRNA levels increased 2- to 4-fold in the spleen and brain after cholesterol feeding for 4 weeks and were higher by 52 to 70% in the spleen of probucol-treated rabbits than in nontreated rabbits. This induction of apoE mRNA occurs within 7 days after the initiation of probucol treatment. However, apoE mRNA levels in the liver, a major apoE-synthesizing organ, were not enhanced after probucol treatment. These results indicate that probucol induces apoE mRNA expression specifically in the spleen and may affect apoE-mediated lipoprotein metabolism, the so-called reverse cholesterol transport.
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As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, respectively. The resultant plasmids directed the expression of 57-kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl beta-D-thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure-function relationships in seed proteins using site-directed mutagenesis. This is the first expression of glycinin-like storage protein in E. coli.
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A cDNA clone covering the entire coding region for a glutelin subunit precursor has been identified from a library of endosperm-developing rice cDNA clones using a mixed oligodeoxynucleotide probe, and then by immunoprecipitation of hybrid-selected translation product with an antiserum against the acidic polypeptides of the glutelin. Analysis of the cDNA insert revealed that rice glutelin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs, like glycinin precursors of soybean. By comparing the predicted protein sequence of this precursor from monocots with that of glycinin A1aB1b precursor from dicots, it was found that the overall 32% of the amino acid positions are identical in both proteins. Because regions which show identities are dispersed throughout both molecules, the similarity is not due to convergent evolution, but to divergence evolution from a common ancestral gene.
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A cDNA clone covering the whole coding region for a glycinin subunit precursor containing the A1a acidic subunit, one of the A2 family, has been identified from a library of soybean cotyledonary cDNA clones using a mixed oligonucleotide probe. Analysis of the cDNA insert revealed that it contained 1746 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 54 nucleotides, a signal peptide region corresponding to 19 amino acids, an acidic subunit region (A1a) corresponding to 291 amino acids followed by a basic subunit region corresponding to 185 amino acids, and a 3'-terminal nontranslated region of 207 nucleotides. By comparing the predicted protein sequence of this precursor with that of the legumin A precursor of pea, it was found that glycinin A2 subunit family appeared to be more closely related to the legumin than to the A3 subunit family, and that the evolutional rearrangement of glycinin genes has occurred.
The complete nucleotide sequence of a cloned cDNA, designated pGA5A4B3822, corresponding to glycinin A5A4B3 mRNA was determined. Analysis of the cDNA insert revealed that it contained 1899 nucleotides of mRNA sequence with a 5'-terminal non-translated region of 31 nucleotides, a signal peptide region corresponding to 23 amino acids, an acidic subunit region (A5) corresponding to 97 amino acids, an acidic subunit region (A4) corresponding to 257 amino acids followed by a basic subunit region (B3) corresponding to 185 amino acids, and a 3'-terminal non-translated region of 182 nucleotides. These results show that the glycinin A4 subunit, which is not found to be linked to a basic subunit via a disulfide bond, is synthesized as a full-sized precursor, i.e. the A5A4B3 subunit complex, from a single mRNA, followed by post-translational processing to generate an intermediary subunit complex (A5-B3), covalently linked by a disulfide bond, and the mature A4 subunit, which may associate with the above subunit complex by non-covalent interactions. From the results obtained by the Chou-Fasman rules we speculated that the two post-translational cleavage sites of this subunit precursor might be processed by the same proteolytic enzyme.
The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.
The amino acid sequence of the A3 subunit, an acidic subunit of the glycinin seed storage protein of the soybean (Glycine max (L.) Merrill), has been determined by sequence analysis of the peptides obtained by cyanogen bromide cleavage followed by enzymatic digestion and partial acid hydrolysis. The A3 subunit is composed of 410 amino acids (Mr approximately 46,000). It has been confirmed that there are conserved, repeated domains within the subunit. A comparison of the NH2-terminal sequence of the A4 subunit with the sequence of the A3 subunit reveals that a sequence highly homologous to the NH2-terminal sequence of the A4 subunit exists in internal regions of the A3 subunit. This suggests that the genes coding for the acidic subunits of glycinin are strongly related, and have the same evolutionary origin.
A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.
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