Identification of the bile acid-binding region in the soy glycinin A1aB1b subunit

Soy glycinin has five major subunits which are classified into two groups according to their homology in amino acid sequences (group I, A1aB1b, A1bB2 and A2B1a; group II, A3B4 and A5A4B3). It has been reported that the peptide fragments derived from the A1a and A2 chains of the A1aB1b and A2B1a subunits had bile acid-binding ability and that the region of 114-161 residues of the A1a chain was responsible for this bile acid-binding ability. In this study, we constructed A1a, A3 and 9 deletion mutants of A1a lacking various numbers of residues at the C-terminus, and evaluated their bile acid-binding ability by a cholic acid-conjugated column and fluorescence analysis. The bile acid-binding ability of A1a was higher than that of A3 and there was a remarkable decrease in the bile acid-binding ability between the delta[138-291] and delta[130-291] mutants. The 130-138 region is rich in hydrophobic residues. In this regard, when we constructed the delta[129-134] mutant lacking six contiguous hydrophobic residues (VAWWMY) and evaluated its bile acid-binding ability, a similar remarkable decrease in the bile acid-binding ability was observed. These results indicate that the 129-134 residue region (VAWWMY) with high hydrophobicity was important for bile acid-binding of A1a.     

Improved Bile Acid-binding Ability of Soybean Glycinin A1a Polypeptide by the Introduction of a Bile Acid-binding Peptide (VAWWMY)

We have previously identified a potential bile acid-binding peptide sequence (VAWWMY) in acidic polypeptide A1a of the soybean glycinin A1aB1b subunit (Choi, S. K., et al., Biosci. Biotechnol. Biochem., 66, 2395–2401 (2002)). In this study, we introduced the nucleotide sequence encoding this peptide in the coding DNA which corresponds to amino acids between 251 and 256, and 282 and 287 into the A1a polypeptide by replacement to respectively give modified versions A1aM1 and A1aM2. A fluorescence analysis demonstrates that their bile acid-binding ability was improved compared to A1a. Moreover, modified proglycinin A1aB1b with the VAWWMY sequence at the same sites as those of A1aM1 and A1aM2 was judged to assume the correct conformation. These results suggest the possibility of developing transgenic crops to accumulate the modified glycinin.     

A cDNA clone encoding a glycinin A1a subunit precursor of soybean.

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.     

Activation of aldo-keto reductase family member 1B14 (AKR1B14) by bile acids: Activation mechanism and bile acid-binding site

Aldo-keto reductase (AKR) 1B14, a rat ortholog of mouse androgen-dependent vas deferens protein (AKR1B7), is involved in the synthesis of prostaglandin F_2α and detoxification of 4-oxononenal formed by lipid peroxidation. The NADPH-linked reductase activity of AKR1B14 was activated by various bile acids. Although the activation was increased by decreasing pH from 9.0 to 6.0, the concentrations giving maximum stimulation (2- to 18-fold) were 0.2-6.0 μM for bile acids at pH 7.4. Kinetic analyses of the activation by glycochenodeoxycholic acid in the forward and reverse reactions, together with fluorescence changes and protection against 4-oxononenal-induced inactivation by bile acid, indicate that the bile acid binds to the enzyme and its coenzyme binary complex as a non-essential activator. The bile acid binding to AKR1B14 mainly accelerates the NADP⁺ dissociation, the rate-limited step of the enzyme reaction. AKR1B7 was also activated by bile acids, but the activation was low and independent of pH. The mutagenesis of His269 and Leu267 of AKR1B14 into the corresponding residues (Arg and Pro, respectively) of AKR1B7 resulted in low and pH-independent activation by bile acids. The results, together with the docking of the bile acid in the recently determined crystal structure of AKR1B14, identify the bile acid-binding site of which His269 plays a key role in significant activation through its electrostatic interaction with the carboxyl group of bile acid, facilitating the release of NADP⁺.     

Ultrastructural localization of glycinin and beta-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

beta-Conglycinin (7 S globulin) and glycinin (11 S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7 S globulin could not be detected.     

Nucleotide sequences encoding glycinin B4 polypeptide of the cultivated soybean Glycine max and its presumed wild-type ancestor Glycine soja in connection with the origin of cultivated soybean

Nucleotide sequences of cDNAs encoding soybean glycinin B4 polypeptide were compared in three soybean cultivars and two plant introductions of wild soybean Glycine soja. Only two nucleotide substitutions were found in three cultivars G. max, as compared with G. max and G. soja having nucleotide sequences which contain four nucleotide substitutions. These data serve as additional evidence, at molecular level, indicating the origin of G. max from G. soja. On the other hand, the time period required for four nucleotide substitutions' accumulation, as calculated from parameters of molecular evolution of 11S globulins, is much longer than the term having passed after soybean domestication.     

Amino acid sequence of a soybean (Glycine max) seed polypeptide having a poly(L-aspartic acid) structure

A polypeptide of Mr 4400 was isolated from soybean (Glycine max) seeds by extraction with 60% ethanol followed by ion-exchange and reverse-phase chromatography. The peptide contains an unusually high amount of aspartic acid residues (approximately 25%, and hence is designated as soybean aspartic acid-rich peptide). Its complete primary structure was determined by conventional methods to be the following. Ser-Lys-Trp-Gln-His-Gln-Gln-Asp-Ser-Cys-Arg-Lys-Gln-Leu-Gln-Gly-Val-Asn- Leu-Thr-Pro-Cys-Glu-Lys-His-Ile-Met-Glu-Lys-Ile-Gln-Gly-Arg-Gly-Asp-Asp- Asp-Asp-Asp-Asp-Asp-Asp-Asp A striking features of this primary structure is the presence of a poly(L-aspartic acid) sequence at the carboxyl terminus, and this polyaspartyl segment was found to precipitate bovine trypsin. The presence of the putative cell attachment sequence Arg-Gly-Asp was also noted.     

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.     

Ethnicity-dependent polymorphism in Na+-taurocholate cotransporting polypeptide (SLC10A1) reveals a domain critical for bile acid substrate recognition

The key transporter responsible for hepatic uptake of bile acids from portal circulation is Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1). This transporter is thought to be critical for the maintenance of enterohepatic recirculation of bile acids and hepatocyte function. Therefore, functionally relevant polymorphisms in this transporter would be predicted to have an important impact on bile acid homeostasis/liver function. However, little is known regarding genetic heterogeneity in NTCP. In this study, we demonstrate the presence of multiple single nucleotide polymorphisms in NTCP in populations of European, African, Chinese, and Hispanic Americans. Specifically four nonsynonymous single nucleotide polymorphisms associated with a significant loss of transport function were identified. Cell surface biotinylation experiments indicated that the altered transport activity of T668C (Ile223-->Thr), a variant seen only in African Americans, was due at least in part to decreased plasma membrane expression. Similar expression patterns were observed when the variant alleles were expressed in HepG2 cells, and plasma membrane expression was assessed using immunofluorescence confocal microscopy. Interestingly the C800T (Ser267-->Phe) variant, seen only in Chinese Americans, exhibited a near complete loss of function for bile acid uptake yet fully normal transport function for the non-bile acid substrate estrone sulfate, suggesting this position may be part of a region in the transporter critical and specific for bile acid substrate recognition. Accordingly, our study indicates functionally important polymorphisms in NTCP exist and that the likelihood of being carriers of such polymorphisms is dependent on ethnicity.     

Identification of a plant introduction soybean line with genetic lesions affecting two distinct glycinin subunits and evaluation of impacts on protein content and composition

Unlike other oilseeds, soybean (Glycine max [L.] Merr) is also valuable due to its direct conversion into human food. One notable example is the cheese-like product tofu. The quality of tofu is improved when protein subunits derived from two glycinin genes, Gy1 and Gy4, are reduced or absent. Here we report the discovery that one exotic soybean plant introduction line, PI 605781 B, has not only a previously described loss-of-expression mutation affecting one glycinin gene (gy4), but also bears an extremely rare, potentially unique, frameshift mutation in the Glycinin1 gene (gy1-a). We analyzed glycinin gene expression via qRT-PCR with mRNA from developing seeds, which revealed that the novel allele dramatically reduced Gy1 mRNA accumulation. Similarly, both A₄A₅B₃ and A_1aB_1a protein subunits were absent or at undetectable levels, as determined by two-dimensional protein fractionation. Despite the reduction in glycinin content, overall seed protein levels were unaffected. The novel gy1-a allele was found to be unique to PI 605871B in a sampling of 247 diverse germplasm lines drawn from a variety of geographic origins.     

Production of an antibody to arterial fatty acid-binding protein (FABP) using a synthetic peptide as the antigen.

1. A procedure is described for the preparation of an antibody to arterial FABP using a synthetic peptide as an antigen. In order to locate a highly conserved region located on the outer surface of FABP, computer analysis of primary and secondary structures of several proteins from the FABP family was undertaken and a 24 amino acid sequence beginning at the fifth position from the N-terminus of rat heart FABP was chosen. 2. The synthetic peptide consisted of eight replications of the 24 amino acid sequence individually attached to the alpha and epsilon amino groups of each terminal lysine on an octalysine branched peptide. 3. Antibody to the synthetic antigen was raised in New Zealand rabbits. Western analysis was conducted and detection was accomplished by using goat-anti-rabbit second antibody conjugated to alkaline phosphatase. 4. The antibody produced from the previously described peptide, recognized purified rat heart FABP and demonstrated a high positive correlation (r = 0.96) when known concentrations of purified hFABP were plotted against densitometric measurement of the bands. 5. Additionally, the antibody recognized FABP from the 104,000 g supernates of rat atrial and arterial tissue fractionated by a Sephadex G-75 column. 6. Therefore, the antibody produced from this particular protocol employing a synthetic peptide can be utilized qualitatively and quantitatively in the analysis of the heart and arterial FABP content.     

The amino acid sequence of the A2B1a subunit of glycinin

The amino acid sequences of the acidic and basic components of the A2B1a subunit of glycinin, the major seed reserve protein of the soybean (Glycine max L. Merr.), were determined. They contain 278 and 180 amino acids, respectively, and have molecular weights of 31,600 +/- 100 and 19,900 +/- 100. The molecular weight of the acidic component is considerably less than that estimated by sodium dodecyl sulfate-gel electrophoresis (37,000). Sequence heterogeneity was detected at several positions scattered throughout the primary structures of both components, indicating that the preparation sequenced was composed of several nearly identical polypeptides. These data, in conjunction with a recently determined nucleotide sequence of the 3'-terminal two-thirds of the analogous glycinin subunit gene, illustrate the complexity of the gene family responsible for synthesis of glycinin subunits.     

A single amino acid mutation in zebrafish (Danio rerio) liver bile acid-binding protein can change the stoichiometry of ligand binding

In all of the liver bile acid-binding proteins (L-BABPs) studied so far, it has been found that the stoichiometry of binding is of two cholate molecules per internal binding site. In this paper, we describe the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) L-BABP to 1.5A resolution, which is currently the highest available for a protein of this family. Since we have found that in zebrafish, the stoichiometry of binding in the protein cavity is of only one cholate molecule per wild type L-BABP, we examined the role of two crucial amino acids present in the binding site. Using site-directed mutagenesis, we have prepared, crystallized, and determined the three-dimensional structure of co-crystals of two mutants. The mutant G55R has the same stoichiometry of binding as the wild type protein, whereas the C91T mutant changes the stoichiometry of binding from one to two ligand molecules in the cavity and therefore appears to be more similar to the other members of the L-BABP family. Based on the presence or absence of a single disulfide bridge, it can be postulated that fish should bind a single cholate molecule, whereas amphibians and higher vertebrates should bind two. Isothermal titration calorimetry has also revealed the presence in the wild type protein and the G55R mutant of an additional binding site, different from the first and probably located on the surface of the molecule.     

A complete cDNA coding for the sequence of glycinin A2B1a subunit precursor

Analysis of the A₂B_1a subunit precursor, one of the A₂-subunit family of glycinin, the main storage protein of soybean, revealed that it is composed of a signal peptide segment (18 amino acids), the A₂ acidic polypeptide (282 amino acids), followed by the B_1a basic polypeptide (185 amino acids). There was overall 63% homology between this subunit complex and pea legumin, which is an analogous protein to glycinin. As this degree of homology is rather higher than that in the A₃B₄ subunit, one of the A₃ subunit family, it seems that the genes encoding the A₂ subunit family are phylogenetically more strongly related to the legumin genes.     

The origin and evolution of peptide YY (PYY) and pancreatic polypeptide (PP)

It is generally accepted that the neuropeptide Y (NPY) family of homologous peptides arose as a result of a series of gene duplication events. Recent advances in comparative genomics allow to formulate a hypothesis that explains, at least in part, the complexity of the family. Chromosome mapping studies reveal that the gene encoding PYY may have arisen from a common ancestral gene (termed NYY) in an ancient chromosomal duplication event that also involved the hox gene clusters. A tandem duplication of the PYY gene concomitant with or just before the emergence of tetrapods generated the PPY gene encoding PP. In the primate and ungulate lineages, the PYY-PPY gene cluster has undergone a more recent gene duplication event to create a PYY2-PPY2 gene cluster on the same chromosome. In the human and baboon, this cluster probably does not encode functional NPY family peptides but expression of the bovine PYY2 gene generates seminalplasmin, a major biologically active component of bull semen. An independent duplication of the PYY gene in the lineage of teleost fish has generated peptides of the PY family that are synthesized in the pancreatic islets of Acanthomorpha. The structural organization of the biosynthetic precursors of PYY and PP (preproPYY and preproPP) has been quite well preserved during the evolution of vertebrates but conservative pressure on individual domains in the proteins has not been uniform. The duplication of the PYY gene that generated the PPY gene appears to have resulted in a relaxation of conservative pressure on the functional domain with the result that the amino acid sequences of tetrapod PYYs are more variable than the PYYs of jawed fish. Although the primary structure of PP has been quite strongly conserved in mammals, with the exception of the rodents, the extreme variability in the sequences of amphibian and reptilian PPs means that the peptide is a useful molecular marker to study the branching order in early tetrapod evolution     

A novel 57 kDa peroxisomal membrane polypeptide detected by monoclonal antibody (PXM1a/207B)

BALB/c mice were immunized with peroxisomal membranes prepared from rat liver. Spleen cells were fused with myeloma cells (P3/U1) and the hybridomas were selected using peroxisomal membranes. A monoclonal antibody (PXM1a/207B) which recognized peroxisomal membranes was selected. Using the antibody, a novel 57 kDa polypeptide was identified in the peroxisomal membrane fraction. Immunoblot analysis of the subcellular fractions demonstrated that the 57 kDa polypeptide was present exclusively in peroxisomal membranes. The 57 kDa polypeptide was partially digested by trypsin and chymotrypsin under conditions where peroxisomal particles remained intact, indicating that the polypeptide is exposed to the cytosolic face of the peroxisomal membrane. The amount of 57 kDa polypeptide increased in parallel with proliferation of peroxisomes by administration of clofibrate.