Prion proteins from susceptible and resistant sheep exhibit some distinct cell biological features

It is well established that natural polymorphisms in the coding sequence of the PrP protein can control the expression of prion disease. Studies with a cell model of sheep prion infection have shown that ovine PrP allele associated with resistance to sheep scrapie may confer resistance by impairing the multiplication of the infectious agent. To further explore the biochemical and cellular mechanisms underlying the genetic control of scrapie susceptibility, we established permissive cells expressing two different PrP variants. In this study, we show that PrP variants with opposite effects on prion multiplication exhibit distinct cell biological features. These findings indicate that cell biological properties of ovine PrP can vary with natural polymorphisms and raise the possibility that differential interactions of PrP variants with the cellular machinery may contribute to permissiveness or resistance to prion multiplication.     

棉铃虫幼虫中肠非糖基磷酯酰肌醇锚着氨肽酶N基因的克隆和测序

通过对棉铃虫Helicoverpa armigera (Hübner)幼虫中肠氨肽酶N的克隆和测序,鉴定了1个氨肽酶N基因APN1,其cDNA序列具有3 220个核苷酸,具有3 042 bp的开放阅读框,编码产生1 014个氨基酸的蛋白质。其推定的氨基酸序列具有氨肽酶N所共有的锌结合模体HEXXHX₁₈E和N末端20个氨基酸的疏水性信号序列,但C末端没有糖基磷酯酰肌醇（glycosylphosphatidylinositol,GPI）锚添加信号序列。该氨肽酶N的cDNA序列已提交GenBank,登录号为AY358034。     

Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells

A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. (c) 1993 John Wiley & Sons, Inc.     

Function of a membrane-embedded domain evolutionarily multiplied in the GPI lipid anchor pathway proteins PIG-B,PIG-M,PIG-U,PIG-W,PIG-V,and PIG-Z

Distant homology relationships among proteins with many transmembrane regions (TMs) are difficult to detect as they are clouded by the TMs’ hydrophobic compositional bias and mutational divergence in connecting loops. In the case of several GPI lipid anchor biosynthesis pathway components, the hidden evolutionary signal can be revealed with dissectHMMER, a sequence similarity search tool focusing on fold-critical, high complexity sequence segments. We find that a sequence module with 10 TMs in PIG-W, described as acyl transferase, is homologous to PIG-U, a transamidase subunit without characterized molecular function, and to mannosyltransferases PIG-B, PIG-M, PIG-V and PIG-Z. We conclude that this new, membrane-embedded domain named BindGPILA functions as the unit for recognizing, binding and stabilizing the GPI lipid anchor in a modification-competent form as this appears the only functional aspect shared among all proteins. Thus, PIG-U's likely molecular function is shuttling/presenting the anchor in a productive conformation to the transamidase complex.     

The Lmgpi15 gene, encoding a component of the glycosylphosphatidylinositol anchor biosynthesis pathway, is required for morphogenesis and pathogenicity in Leptosphaeria maculans

Random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m20, was analysed in detail here. The mutant phenotype was investigated by microscopic analyses of infected plant tissues and in vitro growth assays. Complementation and silencing experiments were used to identify the altered gene. Its function was determined by bioinformatics analyses, cell biology experiments and functional studies. The mutant was blocked at the invasive growth phase after an unaffected initial penetration stage, and displayed a reduced growth rate and an aberrant hyphal morphology in vitro. The T-DNA insertion occurred in the intergenic region between two head-to-tail genes, leading to a complex deregulation of their expression. The unique gene accounting for the mutant phenotype was suggested to be the orthologue of the poorly conserved Saccharomyces cerevisiae gpi15, which encodes for one component of the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway. Consistent with this predicted function, a functional translational fusion with the green fluorescent protein (GFP) was targeted to the endoplasmic reticulum. Moreover, the mutant exhibited an altered cell wall and addition of glucosamine relieved growth defects. It is concluded that the GPI anchor biosynthetic pathway is required for morphogenesis, cell wall integrity and pathogenicity in Leptosphaeria maculans.     

The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish

Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 protein in vitro. Although it has been previously suggested that NS has p53-independent functions, these to date remain unknown. Here, we report two zebrafish mutants recovered from forward and reverse genetic screens that carry loss of function mutations in two members of this nucleolar protein family, Guanine nucleotide binding-protein-like 2 (Gnl2) and Gnl3/NS. We demonstrate that these proteins are required for correct timing of cell cycle exit and subsequent neural differentiation in the brain and retina. Concomitantly, we observe aberrant expression of the cell cycle regulators cyclinD1 and p57kip2. Our models demonstrate that the loss of Gnl2 or NS induces p53 stabilization and p53-mediated apoptosis. However, the retinal differentiation defects are independent of p53 activation. Furthermore, this work demonstrates that Gnl2 and NS have both non-cell autonomously and cell-autonomous function in correct timing of cell cycle exit and neural differentiation. Finally, the data suggest that Gnl2 and NS affect cell cycle exit of neural progenitors by regulating the expression of cell cycle regulators independently of p53.     

Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface

Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) fromBacillus cereus liberated the ghost-linked sialidase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37°C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions). Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4°C for at least 48 h. The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferylN-acetylneuraminic acid (about fourfold), and gave aK _m value of 2.56 · 10^–4 m and an apparentV _max of 2.22 mU per mg protein on GD1a. Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10–12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane. It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface. Abbreviations: PLC, phospholipase C; PIPLC, phospholipase C acting selectively on phosphatidylinositol; NeuAc,N-acetylneuraminic acid; MU, 4-methylumbelliferone; PBS, Dulbecco's phosphate buffer saline solution. Gangliosides were coded according to Svennerholm [42] and the IUPAC-IUB recommendations [43].     

Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form

Transforming growth factor-beta (TGF-beta) is a key modulator of epidermal development and homeostasis, and has been shown to potently regulate keratinocyte migration and function during wound repair. There are three cloned TGF-beta receptors termed type I, type II, and type III that are found on most cell types. The types I and II are the signaling receptors, while the type III is believed to facilitate TGF-beta binding to the types I and II receptors. Recently, we reported that in addition to these receptors, human keratinocytes express a 150 kDa TGF-beta 1 binding protein (r150) which forms a heteromeric complex with the TGF-beta signaling receptors. This accessory receptor was described as glycosyl phosphatidylinositol-specific anchored based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). In the present study, we demonstrate that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta 1 with an affinity similar to those of the types I and II TGF-beta signaling receptors. Furthermore, the PIPLC released (soluble) form of this protein is capable of binding TGF-beta 1 independently from the signaling receptors. In addition, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Our observation that r150 interacts with the TGF-beta signaling receptors, together with the finding that the soluble r150 binds TGF-beta 1 suggest that r150 in either its membrane anchored or soluble form may potentiate or antagonize TGF-beta signaling. Elucidating the mechanism by which r150 functions as an accessory molecule in TGF-beta signaling may be critical to understanding the molecular mechanisms underlying the regulation of TGF-beta action in keratinocytes.     

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.     

Identification of a novel estrogen-sulfating cytosolic SULT from zebrafish: molecular cloning, expression, characterization, and ontogeny study

By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Sequence analysis indicated that this zebrafish SULT belongs to the SULT1 cytosolic SULT gene family. The recombinant form of this novel zebrafish SULT, expressed using the pGEX-2TK expression system and purified from transformed BL21 (DE3) Escherichia coli cells, displayed sulfating activities specifically for estrone and 17beta-estradiol among various endogenous compounds tested as substrates. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. This new zebrafish SULT showed dual pH optima, at 6.5 and 10-10.5, with estrone or n-propyl gallate as substrate. Kinetic constants of the sulfation of estrone, 17beta-estradiol, and n-propyl gallate were determined. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish estrogen-sulfating SULT at the beginning of the hatching period during embryogenesis, which continued throughout the larval stage onto maturity.     

Id2a functions to limit Notch pathway activity and thereby influence the transition from proliferation to differentiation of retinoblasts during zebrafish retinogenesis

During vertebrate retinogenesis, the precise balance between retinoblast proliferation and differentiation is spatially and temporally regulated through a number of intrinsic factors and extrinsic signaling pathways. Moreover, there are complex gene regulatory network interactions between these intrinsic factors and extrinsic pathways, which ultimately function to determine when retinoblasts exit the cell cycle and terminally differentiate. We recently uncovered a cell non-autonomous role for the intrinsic HLH factor, Id2a, in regulating retinoblast proliferation and differentiation, with Id2a-deficient retinae containing an abundance of proliferative retinoblasts and an absence of terminally differentiated retinal neurons and glia. Here, we report that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through a novel RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, we identify a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis.     

The unique plant RhoGAPs are dimeric and contain a CRIB motif required for affinity and specificity towards cognate small G proteins

Plant Rho proteins (ROPs) are inactivated by specific GTPase activating proteins, called RopGAPs. Many of these comprise the exclusive combination of a classic, catalytic Arg-containing RhoGAP domain, and a Cdc42/ Rac interactive binding (CRIB) motif which in animal and fungi has been identified in effectors for Cdc42 and Rac1, but never in any GAP protein. Both elements are required for an efficient RopGAP activity. Here, we analyzed the effect of the CRIB motif on the complex formation and the binding reaction with plant and human Rho proteins by using kinetic and equilibrium methods. We show that RopGAP2 from Arabidopsis thaliana dimerizes via its GAP domain and forms a 2:2 complex with ROP. The CRIB effector motif mediates high affinity and specificity in binding. The catalytic Arg in the context of the CRIB motif is inhibitory for binding. The unusually slow association and dissociation reactions suggest a major conformational change whereby the CRIB motif functions as a lid for binding and/or release of ROP. We propose a two-site interaction model where ROP binds to the CRIB motif as described for the human CRIB effectors and to the catalytic GAP domain as described for animal RhoGAPs.     

Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.     

Two major proteins from locust plasma are involved in coagulation and are specifically precipitated by laminarin, a ß-1,3-glucan

Incubation of plasma of the locust Locusta migratoria, with laminarin induced the precipitation of two major proteins with molecular masses of about 260 000 (P260) and 85 000 Da (P85). This precipitation was not observed when other polysaccharides, such as curdlan, dextran, chitin, cellulose or mannan were used. P260 and P85 were purified to homogeneity by a single step on heparin-sepharose chromatography. Since all attempts to separate P260 from P85, other than the use of sodium dodecyl sulfate, were unsuccessful, it is likely that these two molecules form a complex non-covalently associated. Treatment of P260–P85 complex with N-glycosidase F showed that P260 did not appear to be glycosylated whereas 6% of P85 molecular mass was due to N-linked carbohydrates. On the other hand, no change in molecular masses of P260 or P85 was observed once the complex had been treated with lipase. SDS-PAGE and Western blots of plasma and serum stained with blue Coomassie for proteins or with highly specific polysera to P260 or P85, respectively, showed that P260 was only present in plasma and P85 remained in both samples. This indicates that P260 is likely to be one of the most abundant plasma proteins directly involved in the coagulation process in Locusta migratoria. The addition of plasma or P260–P85 complex to a hemocyte lysate supernatant prior to its activation by laminarin induced a lower protease as well as phenoloxidase activity compared with the control. This reduction of activities was not observed in the presence of serum or when P260–P85 complex was added to a fully activated proPO system.     

Alliinase (alliin lyase) from garlic (Alliium sativum) is glycosylated at ASN146 and forms a complex with a garlic mannose-specific lectin

Alliinase (EC 4.4.1.4) catalyses the production of allicin (thio-2-propene-1-sulfinic acid S-allyl ester), a biologically active compound which is also responsible for the characteristic smell of garlic. It was demonstrated that alliinase which contains 5.5–6% of neutral sugars, gives clear PAS-staining, binds to Con A and can form a complex with garlic mannose-specific lectin (ASA). Evidence that the formation of such a complex is mediated by the interaction of the carbohydrate of the glycoprotein enzyme with the lectin was obtained from a radioligand assay which demonstrated the binding of alliinase to ASA and competitive inhibition of this binding by methyl -d-mannoside. ASA I was shown as the lectin mainly present in the complex with alliinase. The results of this study also demonstrate that alliinase is glycosylated at Asn¹⁴⁶ in the sequence Asn¹⁴⁶-Met¹⁴⁷-Thr¹⁴⁸.Abbreviations ASA (Allium sativum agglutini). Garlic mannose specific lectin(s) - PMSF Phenyl methyl sulfonyl fluoride - HPLC High performance liquid chromatography - SDS-PAGE Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - PAS Periodic acid-Schiff reagent stain - CAPS 3-(cyclohexylamino)-1-propanesulfonic acid - TFA Trifluoracetic acid - HEPES N-2-HydroxyethylpiperazineN-2-ethanesulfonic acid - Tricine N-[2-hydroxyl-1,1-bis(hydroxymethyl)]-ethyl glycine - PVDF poly(vinylene difluoride)     

A 36 kDa monomeric protein and its complex with a 10 kDa protein both isolated from bovine aorta are calpactin-like proteins that differ in their Ca2+-dependent calmodulin-binding and actin-severing properties.

Interaction of plasma membrane with the cytoskeleton involves a large number of proteins, among them a 36 kDa protein that was found to be involved in the interaction with actin filaments. We have isolated a 36 kDa protein from bovine aorta as a monomer and in a complex with a 10 kDa protein. Partial amino acid sequence determinations show that the 36 kDa and 10 kDa proteins isolated from bovine aorta are analogous to or identical with corresponding proteins purified from bovine intestine already described by Kristensen, Saris, Hunter, Hicks, Noonan, Glenney & Tack [(1986) Biochemistry 25, 4497-4503]. We report here that the association of the 10 kDa protein with the 36 kDa protein confers specific calmodulin-binding and actin-severing properties on the complex that are not possessed by the 36 kDa monomer alone. These findings suggest that the protein complex could be involved in thin-filament-related structures or could modulate some Ca2+-regulated events mediated by calmodulin.