Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4

Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B₁₂ to the cytoplasm, impairing the vitamin B₁₂-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B₁₂ transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B₁₂ transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B₁₂-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B₁₂ to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.     

LMBD1 Protein Serves as a Specific Adaptor for Insulin Receptor Internalization

Energy homeostasis is crucial for maintaining normally functioning cells; disturbances in this balance often cause various diseases. The limb region 1 (LMBR1) domain containing 1 gene (lmbrd1) encodes the LMBD1 protein that possesses 9 putative transmembrane domains. LMBD1 has been suggested to be involved in the lysosome in aiding the export of cobalamin. In this study, we determined that LMBD1 plays a regulatory role in the plasma membrane. A micro-positron emission tomography analysis showed that a single-allele knock-out of lmbrd1 increased the ¹⁸F-fluorodeoxyglucose uptake in murine hearts. In addition, the knockdown of lmbrd1 resulted in an up-regulated signaling of the insulin receptor (IR) and its downstream signaling molecule, Akt. Confocal and live total internal reflection fluorescence microscopy showed that LMBD1 co-localized and co-internalized with clathrin and the IR, but not with the transferrin receptor. The results of the mutation analysis and phenotypic rescue experiments indicate that LMBD1 interacts with adaptor protein-2 and is involved in the unique clathrin-mediated endocytosis of the IR. LMBD1 selectively interacts with the IR. The knockdown of lmbrd1 attenuated IR endocytosis, resulting in the perturbation of the IR recycling pathway and consequential enhancement of the IR signaling cascade. In summary, LMBD1 plays an imperative role in mediating and regulating the endocytosis of the IR.     

Dose and age‐dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target‐derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF‐dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax^−/− mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA‐positive, and TrkA^−/− TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75^NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose‐ and age‐dependent fashion, mediated by p75^NTR signaling through TrkA expressing axons. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Role of nucleotide excision repair deficiency in intestinal tumorigenesis in multiple intestinal neoplasia (Min) mice

Mice deficient in the Xeroderma pigmentosum group A (Xpa) gene are defective in nucleotide excision repair (NER) and highly susceptible to skin carcinogenesis after dermal exposure to UV light or chemicals. Min (multiple intestinal neoplasia) mice, heterozygous for a germline nonsense mutation in the tumor suppressor gene adenomatous polyposis coli (Apc), develop intestinal tumors spontaneously and show additional intestinal tumors after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In this study, we investigated the impact of loss of XPA function on PhIP-induced intestinal tumorigenesis in F1 offspring of Min/+ (Apc^+/−) mice crossed with Xpa gene-deficient mice. Apc^+/− mice lacking both alleles of Xpa had higher susceptibility towards toxicity of PhIP, higher levels of PhIP–DNA adducts in the middle and distal small intestines, as well as in liver, and a higher number of small intestinal tumors at 11 weeks, compared with Apc^+/− mice with one or two intact Xpa alleles. Localization of tumors was not affected, being highest in middle and distal small intestines in all genotypes. At 11 weeks of age, the number of spontaneous intestinal tumors was not significantly increased by homozygous loss of Xpa, but untreated Apc^+/−/Xpa^−/− mice had significantly shorter life-spans than their XPA-proficient littermates. Heterozygous loss of Xpa did not affect any of the measured end points. In conclusion, the Xpa gene and the NER pathway are involved in repair of bulky PhIP–DNA adducts in the intestines and the liver, and most probably of DNA lesions leading to spontaneous intestinal tumors. These results confirm a role of the NER pathway also in protection against cancer in internal organs, additional to its well-known importance in protection against skin cancer. An effect of Apc^+/− on adduct levels, additional to that of Xpa^−/−, indicates that the truncated APC protein may affect a repair pathway other than NER.     

A frameshift mutation in the cubilin gene (CUBN) in Beagles with Imerslund–Gräsbeck syndrome (selective cobalamin malabsorption)

Mammals are unable to synthesize cobalamin or vitamin B₁₂ and rely on the uptake of dietary cobalamin. The cubam receptor expressed on the intestinal endothelium is required for the uptake of cobalamin from the gut. Cubam is composed of two protein subunits, amnionless and cubilin, which are encoded by the AMN and CUBN genes respectively. Loss‐of‐function mutations in either the AMN or the CUBN gene lead to hereditary selective cobalamin malabsorption or Imerslund–Gräsbeck syndrome (IGS). We investigated Beagles with IGS and resequenced the whole genome of one affected Beagle at 15× coverage. The analysis of the AMN and CUBN candidate genes revealed a homozygous deletion of a single cytosine in exon 8 of the CUBN gene (c.786delC). This deletion leads to a frameshift and early premature stop codon (p.Asp262Glufs*47) and is, thus, predicted to represent a complete loss‐of‐function allele. We tested three IGS‐affected and 89 control Beagles and found perfect association between the IGS phenotype and the CUBN:c.786delC variant. Given the known role of cubilin in cobalamin transport, which has been firmly established in humans and dogs, our data strongly suggest that the CUBN:c.786delC variant is causing IGS in the investigated Beagles.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Allocation of epiblast cells to germ layer derivatives during mouse gastrulation as studied with a retroviral vector

The embryonic ectoderm, or epiblast, is the source of the three primary germ layers that form during gastrulation in the mouse embryo. Previous studies have investigated the fate of epiblast cells in early gastrulation stages using clonal analysis of cell lineage and in late gastrulation stages using transplantation of labeled grafts. In this study, we studied the fate of late gastrulation stage epiblast using a clonal analysis based on a retroviral vector encoding the Escherichia coli lacZ gene. We found that by reducing the volume of viral suspension injected into each embryo, it was possible to achieve single infectious events. Our analysis of 20 embryos singly infected at the late streak stage and 21 at the head fold stage revealed clonal descendants in only a single germ layer in each embryo. These results indicate that allocation of epiblast progenitors to a single germ layer fate has occurred by late gastrulation in mouse embryos. © 1995 Wiley-Liss, Inc.     

Injury‐induced spinal motor neuron apoptosis is preceded by DNA single‐strand breaks and is p53‐ and Bax‐dependent

The mechanisms of injury‐induced apoptosis of neurons within the spinal cord are not understood. We used a model of peripheral nerve‐spinal cord injury in the rat and mouse to induce motor neuron degeneration. In this animal model, unilateral avulsion of the sciatic nerve causes apoptosis of motor neurons. We tested the hypothesis that p53 and Bax regulate this neuronal apoptosis, and that DNA damage is an early upstream signal. Adult mice and rats received unilateral avulsions causing lumbar motor neurons to achieve endstage apoptosis at 7–14 days postlesion. This motor neuron apoptosis is blocked in bax^?/? and p53^?/? mice. Single‐cell gel electrophoresis (comet assay), immunocytochemistry, and quantitative immunogold electron microscopy were used to measure molecular changes in motor neurons during the progression of apoptosis. Injured motor neurons accumulate single‐strand breaks in DNA by 5 days. p53 accumulates in nuclei of motor neurons destined to undergo apoptosis. p53 is functionally activated by 4–5 days postlesion, as revealed by immunodetection of phosphorylated p53. Preapoptotically, Bax translocates to mitochondria, cytochrome c accumulates in the cytoplasm, and caspase‐3 is activated. These results demonstrate that motor neuron apoptosis in the adult spinal cord is controlled by upstream mechanisms involving DNA damage and activation of p53 and downstream mechanisms involving upregulated Bax and cytochrome c and their translocation, accumulation of mitochondria, and activation of caspase‐3. We conclude that adult motor neuron death after nerve avulsion is DNA damage‐induced, p53‐ and Bax‐dependent apoptosis. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 181–197, 2002; DOI 10.1002/neu.10026     

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos

 The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation. Received: 19 October 1998 / Accepted: 16 February 1999     

Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis

Summary: Mouse embryos homozygous for the allele eed^{l7Rn5‐3354SB} of the Polycomb Group gene embryonic ectoderm development (eed) display a gastrulation defect in which epiblast cells move through the streak and form extraembryonic mesoderm derivatives at the expense of development of the embryo proper. Here we demonstrate that homozygous mutant ES cells have the capacity to differentiate embryonic cell types both in vitro as embryoid bodies and in vivo as chimeric embryos. In chimeric embryos, eed mutant cells can respond to wild‐type signals and participate in normal gastrulation movements. These results indicate a non–cell‐autonomous function for eed. Evidence of mutant cell exclusion from the forebrain and segregation within somites, however, suggests that eed has cell‐autonomous roles in aspects of organogenesis. A requirement for eed in the epiblast during embryonic development is supported by the fact that high‐contribution chimeras could not be rescued by a wild‐type extraembryonic environment. genesis 31:142–146, 2001. © 2001 Wiley‐Liss, Inc.     

Generation of a conditional mouse model to target Acvr1b disruption in adult tissues

Alk4 is a type I receptor that belongs to the transforming growth factor‐beta (TGF‐β) family. It takes part in the signaling of TGF‐β ligands such as Activins, Gdfs, and Nodal that had been demonstrated to participate in numerous mechanisms ranging from early embryonic development to adult‐tissue homeostasis. Evidences indicate that Alk4 is a key regulator of many embryonic processes, but little is known about its signaling in adult tissues and in pathological conditions where Alk4 mutations had been reported. Conventional deletion of Alk4 gene (Acvr1b) results in early embryonic lethality prior gastrulation, which has precluded study of Alk4 functions in postnatal and adult mice. To circumvent this problem, we have generated a conditional Acvr1b floxed‐allele by flanking the fifth and sixth exons of the Acvr1b gene with loxP sites. Cre‐mediated deletion of the floxed allele generates a deleted allele, which behaves as an Acvr1b null allele leading to embryonic lethality in homozygous mutant animals. A tamoxifen‐inducible approach to target disruption of Acvr1b specifically in adult tissues was used and proved to be efficient for studying Alk4 functions in various organs. We report, therefore, a novel conditional model allowing investigation of biological role played by Alk4 in a variety of tissue‐specific contexts. genesis 51:120–127, 2013. © 2012 Wiley Periodicals, Inc.     

Dynamic regulation of LARG in blastopore closure and archenteron formation during Xenopus laevis gastrulation

Rho GTPases have important roles in regulating cell migration and are activated by Rho-specific guanine nucleotide exchange factors (RhoGEFs). However, the role of leukemia-associated RhoGEF (LARG), responding to G12/13 family, has not been studied in vertebrate development. Here, the in vivo biochemical function of LARG was examined during early embryonic development in African frog Xenopus laevis. Gain-of-function study was performed by injecting the RNA of full-length xLARG to 2 cell-stage embryos. The ectopic expression of this protein resulted in the defect of blastopore closure during early embryogenesis. Expression of the dominant-negative form caused the defect in cell movement and following archenteron formation during late gastrulation, which is represented by the blister formation in the ventral side of the embryos. The phenotype was rescued by co-expressing the mutant with Rho or wild type xLARG, confirming the specificity of the dominant-negative activity of xLARG mutant. In this study, I showed for the first time that the spatiotemporal expression of xLARG is very dynamic and specifically regulated in early Xenopus embryonic development and xLARG may mediate Gα₁₃ signal to activate Rho to exert its function in gastrulation movement and archenteron formation. My results implicate that the dynamic regulation of maternal and zygotic xLARG expression and its biochemical activity is necessary for proper gastrulation.     

Participation of NK1.1+ T Cells in the Rejection of lpr αβT Cells When Bone Marrow Cells of Ipr Mice Are Transplanted into B6 Mice

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6-lpr/lpr mouse origin (i.e., lpr→B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3^? IL-2Rβ⁺ NK cells and IL-2Rβ CD3^int cells (i.e., CD3^int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1⁺ and NK1.1^?, among the IL-2Rβ⁺ CD3^int cells. However, the NK1.1⁺ subset (i.e., NK1.1⁺ T cells) was much more radioresistant, and the majority of CD3^int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6-lpr/lpr mice injected with both BM cells of B6-lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti-NK1.1 mAb or anti-asialoGM₁ antibody to eliminate NK cells alone or both NK cells and NK1.1⁺ T cells. When irradiated B6 mice were pretreated with anti-NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1⁺ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double-negative CD4^?8^? cells, in B6-lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.     

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal. Received: 19 August 1997 / Accepted: 11 December 1997