Age-related learning deficits in transgenic mice expressing the 751-amino acid isoform of human beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta-APP), from which the beta-A4 peptide is derived, is considered to be central to the pathogenesis of Alzheimer disease (AD). Transgenic mice expressing the 751-amino acid isoform of human beta-APP (beta-APP751) have been shown to develop early AD-like histopathology with diffuse deposits of beta-A4 and aberrant tau protein expression in the brain, particularly in the hippocampus, cortex, and amygdala. We now report that beta-APP751 transgenic mice exhibit age-dependent deficits in spatial learning in a water-maze task and in spontaneous alternation in a Y maze. These deficits were mild or absent in 6-month-old transgenic mice but were severe in 12-month-old transgenic mice compared to age-matched wild-type control mice. No other behavioral abnormalities were observed. These mice therefore model the progressive learning and memory impairment that is a cardinal feature of AD. These results provide evidence for a relationship between abnormal expression of beta-APP and cognitive impairments.

The subcellular localization of acetyl-CoA carboxylase 2

Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC 6.4.1.2), ACC1 (Mr = 265 kDa) and ACC2 (Mr = 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1–20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC–green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.

Molecular cloning and characterization of an invertebrate cellular retinoic acid binding protein

We have cloned a cDNA and gene from the tobacco hornworm, Manduca sexta, which is related to the vertebrate cellular retinoic acid binding proteins (CRABPs). CRABPs are members of the superfamily of lipid binding proteins (LBPs) and are thought to mediate the effects of retinoic acid (RA) on morphogenesis, differentiation, and homeostasis. This discovery of a Manduca sexta CRABP (msCRABP) demonstrates the presence of a CRABP in invertebrates. Compared with bovine/murine CRABP I, the deduced amino acid sequence of msCRABP is 71% homologous overall and 88% homologous for the ligand binding pocket. The genomic organization of msCRABP is conserved with other CRABP family members and the larger LBP superfamily. Importantly, the promoter region contains a motif that resembles an RA response element characteristic of the promoter region of most CRABPs analyzed. Three-dimensional molecular modeling based on postulated structural homology with bovine/murine CRABP I shows msCRABP has a ligand binding pocket that can accommodate RA. The existence of an invertebrate CRABP has significant evolutionary implications, suggesting CRABPs appeared during the evolution of the LBP superfamily well before vertebrate/invertebrate divergence, instead of much later in evolution in selected vertebrates.

Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

The gene encoding 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO; EC 1.14.12.4) was cloned by using an oligonucleotide probe corresponding to the N terminus of the enzyme to screen a DNA library of Pseudomonas sp. MA-1. The gene encodes for a protein of 379 amino acid residues corresponding to a molecular mass of 41.7 kDa, the same as that previously estimated for MHPCO. MHPCO was expressed in Escherichia coli and found to have the same properties as the native enzyme from Pseudomonas sp. MA-1. This study shows that MHPCO is a homotetrameric protein with one flavin adenine dinucleotide bound per subunit. Sequence comparison of the enzyme with other hydroxylases reveals regions that are conserved among aromatic flavoprotein hydroxylases.

Isolation of human and mouse genes based on homology to REC2, a recombinational repair gene from the fungus Ustilago maydis

A human and a mouse gene have been isolated based on homology to a recombinational repair gene from the corn smut Ustilago maydis. The new human (h) gene, termed hREC2, bears striking resemblance to several others, including hRAD51 and hLIM15. hREC2 is located on human chromosome 14 at q23–24. The overall amino acid sequence reveals characteristic elements of a RECA-like gene yet harbors an src-like phosphorylation site curiously absent from hRAD51 and hLIM15. Unlike these two relatives, hREC2 is expressed in a wide range of tissues including lung, liver, placenta, pancreas, leukocytes, colon, small intestine, brain, and heart, as well as thymus, prostate, spleen, and uterus. Of greatest interest is that hREC2 is undetectable by reverse transcription-coupled PCR in tissue culture unless the cells are treated by ionizing radiation.

Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanism

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2

The ability to sense orientation relative to gravity requires dense particles, called otoconia, which are localized in the vestibular macular organs. In mammals, otoconia are composed of proteins (otoconins) and calcium carbonate crystals in a calcite lattice. Little is known about the mechanisms that regulate otoconial biosynthesis. To begin to elucidate these mechanisms, we have partially sequenced and cloned the major protein component of murine otoconia, otoconin-90 (OC90). The amino acid sequence identified an orphan chimeric human cDNA. Because of its similarity to secretory phospholipase A2 (sPLA2), this gene was referred to as PLA2-like (PLA2L) and enabled the identification of human Oc90. Partial murine cDNA and genomic clones were isolated and shown to be specifically expressed in the developing mouse otocyst. The mature mouse OC90 is composed of 453 residues and contains two domains homologous to sPLA2. The cloning of Oc90 will allow an examination of the role of this protein in otoconial biosynthesis and in diseases that affect the vestibular system.

A Di-Leucine Sequence and a Cluster of Acidic Amino Acids Are Required for Dynamic Retention in the Endosomal Recycling Compartment of Fibroblasts

Insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase, is dynamically retained within the endosomal compartment of fibroblasts. The characteristics of this dynamic retention are rapid internalization from the plasma membrane and slow recycling back to the cell surface. These specialized trafficking kinetics result in <15% of IRAP on the cell surface at steady state, compared with 35% of the transferrin receptor, another transmembrane protein that traffics between endosomes and the cell surface. Here we demonstrate that a 29-amino acid region of IRAP's cytoplasmic domain (residues 56–84) is necessary and sufficient to promote trafficking characteristic of IRAP. A di-leucine sequence and a cluster of acidic amino acids within this region are essential elements of the motif that slows IRAP recycling. Rapid internalization requires any two of three distinct motifs: M15,16, DED64–66, and LL76,77. The DED and LL sequences are part of the motif that regulates recycling, demonstrating that this motif is bifunctional. In this study we used horseradish peroxidase quenching of fluorescence to demonstrate that IRAP is dynamically retained within the transferrin receptor-containing general endosomal recycling compartment. Therefore, our data demonstrate that motifs similar to those that determine targeting among distinct membrane compartments can also regulate the rate of transport of proteins from endosomal compartments. We propose a model for dynamic retention in which IRAP is transported from the general endosomal recycling compartment in specialized, slowly budding recycling vesicles that are distinct from those that mediate rapid recycling back to the surface (e.g., transferrin receptor-containing transport vesicles). It is likely that the dynamic retention of IRAP is an example of a general mechanism for regulating the distribution of proteins between the surface and interior of cells.

Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor.

The mosquito (Aedes aegypti) vitellogenin receptor (AaVgR) is a large membrane-bound protein (214 kDa when linearized) that mediates internalization of vitellogenin, the major yolk-protein precursor, by oocytes during egg development. We have cloned and sequenced two cDNA fragments encompassing the entire coding region of AaVgR mRNA, to our knowledge the first insect VgR sequence to be reported. The 7.3-kb AaVgR mRNA is present only in female germ-line cells and is abundant in previtellogenic oocytes, suggesting that the AaVgR gene is expressed early in oocyte differentiation. The deduced amino acid sequence predicts a 202.7-kDa protein before posttranslational processing. The AaVgR is a member of the low density lipoprotein receptor superfamily, sharing significant homology with the chicken (Gallus gallus) VgR and particularly the Drosophila melanogaster yolk protein receptor, in spite of a very different ligand for the latter. Distance-based phylogenetic analyses suggest that the insect VgR/yolk protein receptor lineage and the vertebrate VgR/low density lipoprotein receptor lineage diverged before the bifurcation of nematode and deuterostome lines.

Sequence and expression of the murine Hoxd-3 homeobox gene.

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.

RIG-E, a human homolog of the murine Ly-6 family, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell.

In vivo all-trans-retinoic acid (ATRA), a differentiation inducer, is capable of causing clinical remission in about 90% of patients with acute promyelocytic leukemia (APL). The molecular basis for the differentiation of APL cells after treatment with ATRA remains obscure and may involve genes other than the known retinoid nuclear transcription factors. We report here the ATRA-induced gene expression in a cell line (NB4) derived from a patient with APL. By differential display-PCR, we isolated and characterized a novel gene (RIG-E) whose expression is up-regulated by ATRA. The gene is 4.0 kb long, consisting of four exons and three introns, and is localized on human chromosome region 8q24. The deduced amino acid sequence predicts a cell surface protein containing 20 amino acids at the N-terminal end corresponding to a signal peptide and an extracellular sequence containing 111 amino acids. The RIG-E coded protein shares some homology with CD59 and with a number of growth factor receptors. It shares high sequence homology with the murine LY-6 multigene family, whose members are small cysteine-rich proteins differentially expressed in several hematopoietic cell lines and appear to function in signal transduction. It seems that so far RIG-E is the closest human homolog of the LY-6 family. Expression of RIG-E is not restricted to myeloid differentiation, because it is also present in thymocytes and in a number of other tissues at different levels.

The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity.

The murine p53 protein contains two nucleic acid-binding sites, a sequence-specific DNA-binding region localized between amino acid residues 102-290 and a nucleic acid-binding site without sequence specificity that has been localized to residues 364-390. Alternative splicing of mRNA generates two forms of this p53 protein. The normal, or majority, splice form (NSp53) retains its carboxyl-terminal sequence-nonspecific nucleic acid-binding site, which can negatively regulate the sequence-specific DNA-binding site. The alternative splice form of p53 (ASp53) replaces amino acid residues 364-390 with 17 different amino acids. This protein fails to bind nucleic acids nonspecifically and is constitutive for sequence-specific DNA binding. Thus, the binding of nucleic acids at the carboxyl terminus regulates sequence-specific DNA binding by p53. The implications of these findings for the activation of p53 transcriptional activity following DNA damage are discussed.

Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another.

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30–42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.