Three novel repetitive units of reelin.

Reelin is a protein involved in the organization of the developing brain of vertebrates. Reelin is divided into three parts, a F-spondin-like domain, a region including the epitopes of the monoclonal and polyclonal anti-Reelin antibodies, and eight-fold Reelin repeats. Reelin repeats consists of a subrepeat A, an EGF-like domain, and a subrepeat B. We found that the anti-Reelin antibodies' recognition region consists of three repetitive units. The correspondence of the repetitive units with the antigenicity is discussed.

Site-directed removal of N-glycosylation sites in BST-1/CD157: effects on molecular and functional heterogeneity.

Cyclic ADP ribose (cADPR) is a novel second messenger that releases calcium from intracellular calcium stores, but works independently of inositol 1,4,5-trisphosphate. In mammals ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and bone marrow stromal cell antigen 1 (BST-1)/CD157. These enzymes are exposed extracellularly and also possess cADPR hydrolase activity, but an intracellular soluble ADP-ribosyl cyclase has been reported in human T-cells. Previously, a soluble form of BST-1/CD157 (sBST-1), which lacked the glycosylphosphatidylinositol-anchored portion, was expressed by a baculovirus-insect-cell system. In this study, we have purified the sBST-1, and it migrated as two major bands by SDS/PAGE, suggesting that it is post-translationally modified. BST-1 contains four putative N-glycosylation sites. Tunicamycin treatment reduced sBST-1 expression in the culture medium, indicating that N-glycosylation is essential for secretion. Site-directed mutagenesis was performed to generate sBST-1 mutants (N1-N4), each preserving a single N-glycosylation site. N1, N3 and N4 were well secreted into the medium, and were each detected as a single band. Although N3 and N4 retained the ADP-ribosyl cyclase activity, the cADPR-hydrolase activity was retained only in N4. We conclude that N-glycosylation of sBST-1 facilitates the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and enzymic activity. Furthermore a crystal has been obtained using the N4 mutant, but not the wild-type sBST-1. Thus the artificial engineering of N-glycosylation sites could be an effective method to generate homogeneous material for structural studies.

Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor.

The metabotropic glutamate receptors (mGluRs) are key receptors in the modulation of excitatory synaptic transmission in the central nervous system. Here we have determined three different crystal structures of the extracellular ligand-binding region of mGluR1--in a complex with glutamate and in two unliganded forms. They all showed disulphide-linked homodimers, whose 'active' and 'resting' conformations are modulated through the dimeric interface by a packed alpha-helical structure. The bi-lobed protomer architectures flexibly change their domain arrangements to form an 'open' or 'closed' conformation. The structures imply that glutamate binding stabilizes both the 'active' dimer and the 'closed' protomer in dynamic equilibrium. Movements of the four domains in the dimer are likely to affect the separation of the transmembrane and intracellular regions, and thereby activate the receptor. This scheme in the initial receptor activation could be applied generally to G-protein-coupled neurotransmitter receptors that possess extracellular ligand-binding sites.

Homology modeling of human leptin/leptin receptor complex.

Leptin receptor mediates the weight regulatory signal carried by the adipocyte-secreted peptide hormone, leptin. It is important to understand the atomic interactions between leptin and the receptor for the therapeutic applications. However, the structure of leptin receptor has not yet been determined. Leptin shows structural similarity to G-CSF, while leptin receptor is similar in amino acid sequence to G-CSF receptor. Because of the similarity between leptin/leptin receptor complex and G-CSF/G-CSF receptor complex, we tried to build a model structure of leptin/leptin receptor complex with the crystal structure of the G-CSF/G-CSF receptor complex as the template. The obtained model for the complex was consistent with the results of the amino acid replacement and deletion experiments. The observation suggests that the model is useful to lead the experimental study on the interaction between leptin and the receptor.

Cryptic dimer interface and domain organization of the extracellular region of metabotropic glutamate receptor subtype 1.

Previously, we produced the whole extracellular region of metabotropic glutamate receptor subtype 1 (mGluR1) in a soluble form. The soluble receptor retained a ligand affinity comparable with that of the full-length membrane-bound receptor and formed a disulfide-linked dimer. Here, we have identified a cysteine residue responsible for the intermolecular disulfide bond and determined domain organization of the extracellular region of mGluR1. A mutant, C140A, was a monomer under nonreduced conditions by SDS-polyacrylamide gel electrophoresis; however, C140A was eluted at the position similar to that of mGluR113, the wild type soluble receptor, by size exclusion column chromatography. Furthermore, C140A bound a ligand, [(3)H]quisqualate, with an affinity similar to that obtained by mGluR113. Oocytes injected with RNA for full-length mGluR1 containing C140A mutation showed responses to ligands at magnitudes similar to those with wild type full-length RNA. Thus, elimination of the disulfide linkage did not perturb the dimer formation and ligand signaling, suggesting that cryptic dimer interface(s) possibly exist in mGluR1. Limited proteolysis of the whole extracellular fragment (residue 33-592) revealed two trypsin-sensitive sites, after the residues Arg(139) and Arg(521). A 15-kDa NH(2)-terminal proteolytic fragment (residue 33-139) was associated with the downstream part after the digestion. Arg(521) was located before a cysteine-rich stretch preceding the transmembrane region. A new shorter soluble receptor (residue 33-522) lacking the cysteine-rich region was designed based on the protease-sensitive boundary. The purified receptor protein gave a K(d) value of 58.1 +/- 0.84 nm, which is compatible to a reported value of the full-length receptor. The B(max) value was 7.06 +/- 0. 82 nmol/mg of protein. These results indicated that the ligand-binding specificity of mGluR1 is confined to the NH(2)-terminal 490-amino acid region of the mature protein.

Recognition of a TG mismatch: the crystal structure of very short patch repair endonuclease in complex with a DNA duplex.

The crystal structure of very short patch repair (Vsr) endonuclease, in complex with Mg2+ and with duplex DNA containing a TG mismatch, has been determined at 2.3 A resolution. In E. coli, the enzyme recognizes a TG mismatched base pair, generated after spontaneous deamination of methylated cytosines, and cleaves the phosphate backbone on the 5' side of the thymine. Extensive interactions between the DNA and the protein characterize a novel recognition mechanism, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking. With the presence of a cleaved DNA intermediate in the active center, the structure of the Vsr/DNA complex provides detailed insights into the catalytic mechanism for endonuclease activity.

Inhibitor peptide SNP-1 binds to a soluble form of BST-1/CD157 at a 2:2 stoichiometry.

Recently we have identified a 15-mer peptide, SNP-1, by a random phage library that can bind to bone marrow stromal cell antigen-1 (BST-1)/CD157 [Sato, A., Yamamoto, S., Ishihara, K., Hirano, T. & Jingami, H. (1999) Biochem. J. 337, 491-496]. SNP-1 inhibits BST-1 ADP-ribosyl cyclase activity uncompetitively with a Ki value of 180 +/- 40 nM. In this study we analysed biophysically the SNP-1 binding to a soluble form of BST-1 (sBST-1). Equilibrium binding data of wild-type SNP-1 from surface plasmon resonance studies gave a Kd value of 500 +/- 35 nM. Titration calorimetry analysis showed that the binding reaction is exothermic at 20 degrees C. The values of Kd = 211 nM, enthalpy change, DeltaH = -18.68 kcal.mol-1, and saturated molar ratio of bound SNP-1 per sBST-1, N = 0.8 mol.mol-1 were obtained. On the basis of the molecular masses of SNP-1 and sBST-1 calculated by analytical ultracentrifugation, the stoichiometry of the binding was determined to be 2 : 2. Electron microscopy also revealed the dimer form of sBST-1. To delineate the core residue of SNP-1 responsible for binding, each amino acid residue has been replaced by alanine. A region from amino acid residues 7-12 appeared to be critical for the SNP-1 binding to sBST-1. The substitution of the first residue, His, to Ala led to a reduction in binding, suggesting that the N-terminal residue is also crucial.

39-kDa receptor-associated protein (RAP) facilitates secretion and ligand binding of extracellular region of very-low-density-lipoprotein receptor: implications for a distinct pathway from low-density-lipoprotein receptor.

We have expressed the extracellular regions of the low-density-lipoprotein (LDL) receptor and the very-low-density-lipoprotein (VLDL) receptor, along with the full-length forms of the receptors, in insect cells in a baculovirus system. The extracellular region of the LDL receptor has been secreted successfully into the culture medium, and it retained the capacities of binding 125I-labelled LDL and beta-VLDL. In contrast, the extracellular region of the VLDL receptor remained intracellular and it did not bind 125I-beta-VLDL. This difference in expression behaviour between the homologous regions of the two receptors suggests that the two receptor systems are different in receptor-protein maturation or protein targeting. Next we developed the co-expression system with 39-kDa receptor-associated protein (RAP). This co-expression facilitated the secretion of the extracellular region of the VLDL receptor into the culture medium and the secreted receptor bound 125I-beta-VLDL. The VLDL receptor remaining intracellular that was co-expressed with RAP also showed binding capacity to 125I-beta-VLDL, implying that the existence of RAP prevented receptor-protein aggregation or improved protein-folding status of the truncated VLDL receptor. On the other hand, expression of the extracellular region of the LDL receptor was not facilitated by RAP co-expression. Thus RAP plays an essential role in maintenance of the active conformation and secretion of the extracellular region of the VLDL receptor.

Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1.

N-Methyl-D-aspartate receptor channels are composed of an NR1 subunit and at least one of the NR2 subunits (NR2A-D). Activation of the N-methyl-d-aspartate receptor requires the co-agonists glycine and glutamate. It has been proposed that the NR1 subunit possesses a glycine-binding site. We have expressed a soluble form of the NR1 subunit, which was produced by connecting the N-terminal extracellular region with the extracellular loop between the third and fourth membrane segments, by a baculovirus system along with full-length and truncated membrane-bound forms. The soluble NR1 receptor was efficiently secreted into the culture medium and showed a high affinity for ligands. The Kd of a glycine-site antagonist, [3H]MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4, 6-dichloro-1H-indole-2-carboxylic acid], for the soluble receptor was 3.89+/-0.97 nM, which was comparable to the Kd of 4.47+/-1.39 nM for the membrane-bound full-length form. These values were close to the values reported previously with the use of rat brain membranes and Chinese hamster ovary cells expressing the full-length form of the NR1 subunit. The Ki values of other glycine-site antagonists, L-689,560 (trans-2-carboxy-5,7-dichloro - 4 - phenylaminocarbonylamino - 1,2,3,4 - tetrahydroquinoline), 5, 7-dichlorokynurenate and 5,7-dinitroquinoxaline-2,3-dione, for the soluble receptor were also similar to those for the full-length form of NR1. [3H]MDL 105,519 binding was also inhibited by the agonists glycine and d-serine. Thus the affinity and selectivity of ligand-binding characteristics of the NR1 subunit is conferred on the soluble form of the NR1 subunit. This soluble receptor provides a good experimental tool for initiating a biophysical analysis of the N-methyl-d-aspartate receptor channel protein.

Crystallographic and functional studies of very short patch repair endonuclease.

Vsr endonuclease plays a crucial role in the repair of TG mismatched base pairs, which are generated by the spontaneous degradation of methylated cytidines; Vsr recognizes the mismatched base pair and cleaves the phosphate backbone 5' to the thymidine. We have determined the crystal structure of a truncated form of this endonuclease at 1.8 A resolution. The protein contains one structural zinc-binding module. Unexpectedly, its overall topology resembles members of the type II restriction endonuclease family. Subsequent mutational and biochemical analyses showed that certain elements in the catalytic site are also conserved. However, the identification of a critical histidine and evidence of an active site metal-binding coordination that is novel to endonucleases indicate a distinct catalytic mechanism.

Novel peptide inhibitor of ecto-ADP-ribosyl cyclase of bone marrow stromal cell antigen-1 (BST-1/CD157).

cADP-ribose, which is produced by ecto-ADP-ribosyl cyclase of CD38 or bone marrow stromal cell antigen-1 (BST-1), is a key regulator of Ca2+-induced calcium release in an Ins(1,4,5)P3-independent manner. In the present study, we have identified a specific peptide inhibitor for ADP-ribosyl cyclase of BST-1. A 15-mer random peptide phage library was screened with a soluble form of BST-1 expressed in baculovirus-infected insect cells. After biopanning, two potent sequences reactive towards BST-1 were isolated. The two synthetic peptides corresponding to the identified sequences were shown to antagonize each other's ability to inhibit the binding of the two isolated phage to BST-1, suggesting that they bind at a common binding site on BST-1. One of the peptides (SNP-1) was shown to inhibit ADP-ribosyl cyclase activity of BST-1 in an uncompetitive manner with a Ki value of 180+/-40 nM (n=3). SNP-1 also inhibited cyclic ADP-ribose hydrolase activity of BST-1 dose-dependently. Selected phage did not cross-react with a soluble form of CD38. SNP-1 did not show any inhibitory effect on ADP-ribosyl cyclase activity of CD38, and therefore this peptide inhibitor will be useful to serve as a starting tool for understanding the roles of these intriguing ecto-enzymes. This is the first report of a specific ADP-ribosyl cyclase inhibitor.

Clinical manifestations due to a point mutation of the mitochondrial tRNAleu(UUR) gene in five families with diabetes mellitus.

It has been shown that an adenine (A) to guanine (G) transition at position 3243 of the mitochondrial transfer RNA(tRNA)leu(UUR) gene is associated with a subgroup of diabetes mellitus. Therefore, we screened for this transition in 86 patients with non-insulin-dependent diabetes mellitus (NIDDM) in which two or three generations were affected with diabetes, in 14 patients with insulin-dependent diabetes mellitus, and in 9 families with diabetes mellitus and/or associated disorders suggesting mitochondrial gene abnormalities. We failed to identify the mutation in 100 diabetic patients, 86 NIDDM and 14 insulin-dependent diabetes mellitus (IDDM). Out of the latter 9 families, we identified an A to G transition in 14 individuals in 5 families. Diabetes mellitus was shown to be maternally inherited in one family. In 9 of 14 patients with the mutation, insulin was required to treat diabetes mellitus, indicating impaired insulin secretion. A hyperglycemic clamp test performed in one subject revealed significant impairment of insulin secretion, whereas euglycemic clamp test showed normal insulin sensitivity in this patient. The heteroplasmy of the mutant mitochondrial DNA (mtDNA) in leukocytes does not appear to correlate with the severity of diabetes in terms of the insulin therapy required. Body mass index of the affected individuals was less than 23.3. In one family, in addition to diabetes mellitus and hearing loss, hypoparathyroidism was associated with the mutation, suggesting that hypoparathyroidism is caused by the impaired processing and/or secretion of proparathyroid hormone due to the mutation. In addition, the affected subjects presented with proteinuria at the time of diagnosis of diabetes mellitus which appeared not to be related with diabetic nephropathy.

Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1.

Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC50) of quisqualate for the soluble receptor was 3.8 x 10(-8) M, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate >> ibotenate >/= L-glutamate approximately (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.

Apolipoprotein E Sendai (arginine 145-->proline): a new variant associated with lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a novel disease characterized by proteinuria, lipoprotein thrombi in the glomeruli, and increased concentration of plasma apolipoprotein (apo) E. It is believed that a genetic disorder of apo E may be present and associated with the disease. Three patients with LPG were examined in this study. The patients' DNA sequences were analyzed, and a nucleotide G to C point mutation in exon 4 of the apo E gene was confirmed in each patient. This missense mutation denotes amino acid substitution of the proline residue for arginine residue at position 145 of apo E. This variant (apo E Sendai) may cause a marked molecular conformational change of the apo E. These findings suggest that a novel variant is etiologically related to LPG.

Effects of gemfibrozil administration on very low density lipoprotein receptor mRNA levels in rabbits.

To elucidate the regulation of very low density lipoprotein (VLDL) receptor gene expression, we administered to rabbits for 14 days gemfibrozil, a fabric acid derivative and a lipid lowering drug that is also included among peroxisome proliferators. VLDL receptor mRNA levels were examined by Northern blot analysis. The VLDL receptor mRNA levels in retroperitoneal adipose tissue and in gastrocnemius muscle were increased 6.9-fold and 3.7-fold, respectively, with gemfibrozil treatment, but no marked changes were observed in the heart, the organ in which VLDL receptor is most highly expressed. In the liver, VLDL receptor mRNA was not detected either before or after gemfibrozil administration. Lipoprotein lipase (LPL) and long-chain acyl coenzyme A synthetase (ACS) mRNA levels were also increased in parallel in adipose tissue. The enhanced expression of VLDL receptor mRNA may contribute to the increase of triglyceride-rich lipoprotein catabolism in peripheral tissues such as adipose tissue and muscles.

Regulation of very-low-density lipoprotein receptor in hypertrophic rat heart.

To elucidate the regulation of very-low density-lipoprotein (VLDL) receptor, we have studied its gene expression in the heart of spontaneously hypertensive rats-stroke prone (SHR-SP, an animal model for hypertension-induced cardiac hypertrophy) compared with Wistar-Kyoto rats. RNase protection assay showed that ventricular VLDL receptor mRNA falls to 41% of normal levels at 4 weeks when hypertension is not yet fully developed, and drops further to 14% at 13 weeks, when cardiac hypertrophy is established. Lipoprotein lipase mRNA decreases in parallel with VLDL receptor mRNA. In cultured neonatal rat ventricular cardiomyocytes, VLDL receptor mRNA decreases in parallel with the process of cardiocyte hypertrophy during the 24 hours after treatment with 10-8 mol/L endothelin-1, falling to 40% of the initial value. These results demonstrate that there is downregulation of VLDL receptor gene expression in cardiac hypertrophy both in vivo and in vitro and suggest that the regulation of the VLDL receptor is possibly linked with the switch in energy substrate from lipid to glucose known to occur in cardiac hypertrophy.

The VLDL receptor: wayward brother of the LDL receptor.

The VLDL receptor binds lipoproteins that contain apolipoprotein E and consists of five functional domains that resemble the LDL receptor. Although the LDL- and VLDL-receptor genes are located on different chromosomes, their gene structure and organization are almost the same. Despite the presence of sterol regulatory element-1-like sequences in the VLDL-receptor gene, the transcription of the gene is not down-regulated by sterols. The VLDL-receptor messenger RNA is highly expressed in tissues that actively metabolize fatty acids as their source of energy. The physiological role of the chicken cognate of the VLDL receptor has been revealed recently, but its function in mammals is still debatable.

Decreased expression of the very low density lipoprotein receptor mRNA in the cardiac ventricle of spontaneously hypertensive rats.

1. To elucidate the functional implication of very low density lipoprotein (VLDL) receptor, we studied the gene expression of VLDL receptor in rats. The VLDL receptor mRNA was highly expressed in the cardiac ventricle and skeletal muscle. Intermediate amounts of VLDL receptor mRNA were detected in adipose tissue, adrenal gland, brain and lung. Thus the tissue distribution of VLDL receptor mRNA in rats was similar to that reported previously in rabbits. 2. We studied the gene expression of the VLDL receptor in the heart of stroke-prone spontaneously hypertensive rats (SHRSP), an animal model for hypertension-induced cardiac hypertrophy. RNase protection assay showed that the level of ventricular VLDL receptor mRNA was already decreased to one half when hypertension was not fully developed, and further diminished to one fifth when cardiac hypertrophy was established. 3. It is reported that energy utilization in SHRSP hypertrophied myocardium is impaired. Our results suggest that inactive fatty acid metabolism in the ventricle of SHRSP is related to the lowered expression of the VLDL receptor which is postulated as a gate for triglyceride-rich lipoprotein particle.

[HMG-CoA reductase inhibitor for therapy of patients with hyperlipoproteinemia ].

The discovery and production of HMGCoA reductase inhibitor and the fundamental research work of the LDL receptor unraveled a receptor-mediated cholesterol homeostasis. HMGCoA reductase inhibitors are the most commonly prescribed class of lipid-lowering drugs in many countries. The decrease of the intracellular cholesterol caused by the inhibitor induces the compensatory increase of LDL receptor protein at liver plasma membrane. The increased receptor promotes LDL catabolism and results in decrease of plasma LDL. Serious side effects involving the liver or muscle are rare. But the risk of myopathy is increased when the drug is used with other hypolipidemic agents. A principle of the treatment of hyperlipidemia, including secondary one associated with diabetes mellitus and renal disease, by HMGCoA reductase is discussed in this review.