scholarly journals Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells

1994 ◽  
Vol 299 (1) ◽  
pp. 171-176 ◽  
Author(s):  
S Howell ◽  
C Lanctôt ◽  
G Boileau ◽  
P Crine
Keyword(s):  
Cell Surface ◽  
Cell Types ◽  
Neutral Endopeptidase ◽  
Integral Membrane Protein ◽  
Cellular Transport ◽  
Type Ii ◽  
Wild Type ◽  
Form Expression ◽  
Km Value ◽  
Frame Fusion

Neutral endopeptidase (EC 3.4.24.11, NEP) is a type-II integral membrane protein found in a wide variety of cell types. We previously produced a secreted form of the enzyme by deletion of the cytoplasmic and transmembrane domains and in-frame fusion of the cleavable signal peptide of pro-opiomelanocortin [Lemay, Waksman, Roques, Crine and Boileau (1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secreted form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-anchor attachment signal of decay-accelerating factor to produce a GPI-anchored form. Expression of this chimeric form in Cos-1 cells resulted in cell-surface activity. This activity could be released from the cell surface by phosphatidylinositol-specific phospholipase C and radiolabelling studies showed that the protein could incorporate [3H]ethanolamine, indicating that the enzyme was GPI-anchored. The Km value, using [D-Ala2,Leu5]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 microM) was comparable with that of wild-type NEP (70 +/- 4 microM), as were the sensitivities to the inhibitors phosphoramidon and thiorphan. However, pulse-chase studies showed that the biosynthesis and cell-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a lower rate of biosynthesis and/or cellular transport for GPI-anchored NEP compared with its transmembrane counterpart.

scholarly journals Efficient endosomal localization of major histocompatibility complex class II-invariant chain complexes requires multimerization of the invariant chain targeting sequence.

1995 ◽  
Vol 129 (5) ◽  
pp. 1217-1228 ◽  
Author(s):  
L S Arneson ◽  
J Miller
Keyword(s):  
Cell Surface ◽  
Class Ii ◽  
Cell Surface Expression ◽  
Invariant Chain ◽  
Antigenic Peptides ◽  
Type Ii ◽  
Wild Type ◽  
Chain Complexes ◽  
Cytosolic Domain ◽  
Localization Signal

During biosynthesis, MHC class II-invariant chain complexes are transported into endosomal compartments where invariant chain (Ii) is degraded and class II encounters antigenic peptides. One of the signals that determines this intracellular transport route has been localized to the cytosolic domain of Ii. Deletion of this signal disrupts endosomal targeting and results in the stable expression of class II-Ii complexes at the surface. In this paper we have examined the role of Ii trimerization on the generation of this endosomal localization signal. In L cell transfectants expressing class II and both wild type Ii and a truncated form of Ii that lacks this endosomal localization signal, Ii was found to form multimers which could contain both wild type and truncated Ii. The multimers were not large aggregates but were found to be discrete complexes, probably the nine molecule class II-Ii complex that has been observed in human B cells. The co-expression of truncated Ii allowed for cell surface expression of a subset of wild type Ii. This surface-expressed wild type Ii associated with truncated Ii in multimers at a 2:1 ratio, indicating that these trimers contain two truncated and one wild type Ii molecule. These data suggest a division in trafficking of Ii trimers: if two wild type Ii molecules are present, the complex is transported to and rapidly degraded in endosomes, whereas the presence of only one wild type Ii results in trafficking and expression of the heterotrimer on the cell surface. Following surface arrival, complexes containing only a single wild type Ii molecule are internalized more rapidly and have a shorter half-life than complexes containing only truncated Ii molecules. These data suggest that although a single Ii cytosolic domain can function as a plasma membrane internalization signal, multimerization of Ii is required for efficient Golgi complex to endosome targeting of class II-Ii complexes.

scholarly journals Secretion of a type II integral membrane protein induced by mutation of the transmembrane segment

1997 ◽  
Vol 322 (1) ◽  
pp. 335-342 ◽  
Author(s):  
Isabelle LEMIRE ◽  
Claude LAZURE ◽  
Philippe CRINE ◽  
Guy BOILEAU
Keyword(s):  
Helical Structure ◽  
Structural Data ◽  
Neutral Endopeptidase ◽  
Integral Membrane Protein ◽  
Full Length ◽  
Signal Peptidase ◽  
Soluble Form ◽  
Type Ii ◽  
Α Helix ◽  
Flanking Regions

Signal peptide/membrane anchor (SA) domains of type II membrane proteins initiate the translocation of downstream polypeptides across the endoplasmic reticulum (ER) membrane. In contrast with signal peptides, however, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncated (without cytoplasmic domain) protein were constructed by substitution of the sequences SQNS, QQTT or YPGY for VTMI starting at position 15 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of these sequences was decided from our previous observation that substitution in the NEP SA domain of the sequence SQNS, which is polar and has α-helix-breaking potential, could promote SA domain processing under certain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268, 2699Ő2704; Yang, Chatellard, Lazure, Crine and Boileau (1994) Arch. Biochem. Biophys. 315, 382Ő386). The QQTT sequence is polar but, according to secondary structure predictions, is compatible with the α-helix structure of the NEP SA domain. The YPGY sequence and single Pro residue are less polar and have α-helix-breaking potential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptides encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-1 cells and their fate (membrane-bound or secreted) was determined by immunoblotting and by endoglycosidase digestions. Our biochemical and structural data indicate that: (1) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) α-helix-breaking residues are not a prerequisite for cleavage; (3) the presence, in close proximity to a putative signal peptidase cleavage site, of a polar sequence that maintains the α-helical structure of the SA domain is sufficient to promote cleavage. Furthermore pulseŐchase studies suggest that cleavage is performed in the ER by signal peptidase and indicate that cleavage is not a limiting step in the biosynthesis of the soluble form of the protein.

scholarly journals Interaction of mammalian neprilysin with binding protein and calnexin in Schizosaccharomyces pombe

1999 ◽  
Vol 340 (3) ◽  
pp. 813-819 ◽  
Author(s):  
Hugues BEAULIEU ◽  
Aram ELAGÖZ ◽  
Philippe CRINE ◽  
Luis A. ROKEACH
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Binding Protein ◽  
Cell Types ◽  
Neutral Endopeptidase ◽  
Integral Membrane Protein ◽  
Eukaryotic Cell ◽  
Unfolded Proteins ◽  
Folding Intermediates ◽  
Bona Fide ◽  
The Brain

Neutral endopeptidase (neprilysin or NEP, EC 3.4.24.11) is a zinc metallo-endopeptidase expressed in many eukaryotic cell types and displaying several important physiological roles. In the brain (and central nervous system), this enzyme is involved in the molecular mechanism of pain by its action in the degradation of enkephalin molecules. In the kidney, NEP is implicated in the degradation of regulatory factors involved in the control of arterial pressure, including atrial natriuretic peptide and bradykinin. In this study we assessed the potential of the fission yeast Schizosaccharomyces pombe to overproduce rabbit NEP and secreted NEP (sNEP, a soluble derivative of this integral membrane protein). Both recombinant NEP and sNEP were produced at high levels (5 mg/l) in this system. Enzymic studies revealed that these recombinant proteins were fully active and exhibit kinetic parameters similar to those of the bona fide enzyme. Immunofluorescence microscopy and enzymic assays demonstrated that recombinant NEP is correctly targeted to the cell membrane. Furthermore, co-immunoprecipitation studies showed that folding intermediates of NEP and sNEP, produced in S. pombe, interact in the endoplasmic reticulum (ER) with binding protein (BiP) and calnexin (Cnx1p). The amount of sNEP coprecipitated with both BiP and Cnx1p augmented when cells were subjected to various stresses causing the accumulation of unfolded proteins in the ER. The interactions of NEP with BiP and Cnx1p were, however, more refractive to the same stresses.

scholarly journals CD23 (Fc∊RII) release from cell membranes is mediated by a membrane-bound metalloprotease

1998 ◽  
Vol 333 (3) ◽  
pp. 573-579 ◽  
Author(s):  
Ariane E. MAROLEWSKI ◽  
Derek R. BUCKLE ◽  
Gary CHRISTIE ◽  
David L. EARNSHAW ◽  
Pearl L. FLAMBERG ◽  
...  
Keyword(s):  
Cell Surface ◽  
Serine Proteases ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Cell Types ◽  
Cysteine Proteases ◽  
Integral Membrane Protein ◽  
Monocytic Cell ◽  
Barr Virus ◽  
Epstein Barr

CD23 (low-affinity IgE receptor, FcεRII) is expressed as a Type II extracellular protein on a variety of cells such as B-cells, monocytes and macrophages and is cleaved from the cell surface to generate several distinct fragments. The expression of CD23 on the cell surface as well as the generation of soluble fragments of CD23 has been shown to be involved in the regulation of IgE synthesis. Here we report that the release of CD23 from the cell surface is mediated by a metalloprotease. An assay utilizing purified CD23 and an neo-epitope antibody specific for one of the known cleavage products is described and used to demonstrate unambiguously the cleavage of CD23 by a distinct protease. Characterization of the mechanism of CD23 processing shows that the protease exists as an integral membrane protein with a functional molecular mass of approx. 63 kDa as determined by gel-filtration chromatography. The CD23-cleaving activity found in enriched plasma membranes from RPMI 8866 cells is inhibited by the metalloprotease inhibitors 1,10-phenanthroline and imidazole and by the matrix metalloprotease inhibitor batimastat, but not by inhibitors of cysteine proteases, serine proteases or acid proteases. The same or a similar activity that cleaves CD23 to the known 33 kDa fragment and is inhibited by batimastat is present in diverse cell types such as unstimulated fibroblasts and monocytic cell lines not expressing CD23, as well as in the Epstein–Barr virus-transformed B-cell line, RPMI 8866, which constitutively expresses CD23.

scholarly journals O-Glycosylation as a Sorting Determinant for Cell Surface Delivery in Yeast

2004 ◽  
Vol 15 (4) ◽  
pp. 1533-1543 ◽  
Author(s):  
Tomasz J. Proszynski ◽  
Kai Simons ◽  
Michel Bagnat
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Membrane Protein ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Chimeric Protein ◽  
Initial Step ◽  
Integral Membrane Protein ◽  
Wild Type

Little is known about the mechanisms that determine localization of proteins to the plasma membrane in Saccharomyces cerevisiae. The length of the transmembrane domains and association of proteins with lipid rafts have been proposed to play a role in sorting to the cell surface. Here, we report that Fus1p, an O-glycosylated integral membrane protein involved in cell fusion during yeast mating, requires O-glycosylation for cell surface delivery. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A chimeric protein lacking O-glycosylation motif was missorted to the vacuole and accumulated in late Golgi in wild-type cells. Exocytosis of this protein could be restored by addition of a 33-amino acid portion of an O-glycosylated sequence from Fus1p. Our data suggest that O-glycosylation functions as a sorting determinant for cell surface delivery of Fus1p.

scholarly journals Intracellular maturation and transport of the SV5 type II glycoprotein hemagglutinin-neuraminidase: specific and transient association with GRP78-BiP in the endoplasmic reticulum and extensive internalization from the cell surface.

1989 ◽  
Vol 109 (6) ◽  
pp. 3273-3289 ◽  
Author(s):  
D T Ng ◽  
R E Randall ◽  
R A Lamb
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Inverse Correlation ◽  
Integral Membrane Protein ◽  
Type Ii ◽  
Sedimentation Analysis ◽  
Specific Antibodies ◽  
Transport Inhibitor ◽  
Infected Cells ◽  
Endocytic Vesicles

The hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus SV5 is a type II integral membrane protein that is expressed at the infected cell surface. The intracellular assembly and transport of HN in CV1 cells was examined using conformation-specific HN mAbs and sucrose density sedimentation analysis. HN was found to oligomerize with a t1/2 of 25-30 min and these data suggest the oligomer is a tetramer consisting primarily of two noncovalently associated disulfide-linked dimers. As HN oligomers could be found that were sensitive to endoglycosidase H digestion and oligomers formed in the presence of the ER to the Golgi complex transport inhibitor, carbonylcyanide m-chlorophenylhydrazone (CCCP), these data are consistent with HN oligomerization occurring in the ER. Unfolded or immature HN molecules that could not be recognized by conformation-specific antibodies were found to specifically associate with the resident ER protein GRP78-BiP. Immunoprecipitation of BiP-HN complexes with an immunoglobulin heavy-chain binding protein (BiP) antibody indicated that newly synthesized HN associated and dissociated from GRP78-BiP (t1/2 20-25 min) in an inverse correlation with the gain in reactivity with a HN conformation-specific antibody, suggesting that the transient association of GRP78-BiP with immature HN is part of the normal HN maturation pathway. After pulse-labeling of HN in infected cells, it was found that HN is rapidly turned over in cells (t1/2 2-2.5 h). This led to the finding that the vast majority of HN expressed at the cell surface, rather than being incorporated into budding virions, is internalized and degraded after localization to endocytic vesicles and lysosomes.

The organization of cell surface membrane domains

1989 ◽  
Vol 47 ◽  
pp. 804-805
Author(s):  
Michael Edidin
Keyword(s):  
Cell Surface ◽  
Membrane Lipids ◽  
Surface Membrane ◽  
Lateral Diffusion ◽  
Cell Types ◽  
Membrane Domains ◽  
Membrane Biology ◽  
Morphological Polarity ◽  
Discrete Domains ◽  
Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

Two Different UGT1A1 Mutations causing Crigler–Najjar Syndrome types I and II in an Iranian Family

2015 ◽  
Vol 24 (4) ◽  
pp. 523-526 ◽  
Author(s):  
Yoshihiro Maruo ◽  
Mahdiyeh Behnam ◽  
Shinichi Ikushiro ◽  
Sayuri Nakahara ◽  
Narges Nouri ◽  
...  
Keyword(s):  
Serum Bilirubin ◽  
Total Serum Bilirubin ◽  
Total Serum ◽  
Compound Heterozygous ◽  
Type I ◽  
Syndrome Type ◽  
Type Ii ◽  
Wild Type ◽  
Iranian Family ◽  
Udp Glucuronosyltransferase

Background: Crigler–Najjar syndrome type I (CN-1) and type II (CN-2) are rare hereditary unconjugated hyperbilirubinemia disorders. However, there have been no reports regarding the co-existence of CN-1 and CN-2 in one family. We experienced a case of an Iranian family that included members with either CN-1 or CN-2. Genetic analysis revealed a mutation in the bilirubin UDP-glucuronosyltransferase (UGT1A1) gene that resulted in residual enzymatic activity.Case report: The female proband developed severe hyperbilirubinemia [total serum bilirubin concentration (TB) = 34.8 mg/dL] with bilirubin encephalopathy (kernicterus) and died after liver transplantation. Her family history included a cousin with kernicterus (TB = 30.0 mg/dL) diagnosed as CN-1. Her great grandfather (TB unknown) and uncle (TB = 23.0 mg/dL) developed jaundice, but without any treatment, they remained healthy as CN-2. Results: The affected cousin was homozygous for a novel frameshift mutation (c.381insGG, p.C127WfsX23). The affected uncle was compound heterozygous for p.C127WfsX23 and p.V225G linked with A(TA)7TAA. p.V225G-UGT1A1 reduced glucuronidation activity to 60% of wild-type. Thus, linkage of A(TA)7TAA and p.V225G might reduce UGT1A1 activity to 18%–36 % of the wild-type. Conclusion: Genetic and in vitro expression analyses are useful for accurate genetic counseling for a family with a history of both CN-1 and CN-2. Abbreviations: CN-1: Crigler–Najjar syndrome type I; CN-2: Crigler–Najjar syndrome type II; GS: Gilbert syndrome; UGT1A1: bilirubin UDP-glucuronosyltransferase; WT: Wild type; TB: total serum bilirubin.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

scholarly journals Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

2016 ◽  
Vol 113 (34) ◽  
pp. E4995-E5004 ◽  
Author(s):  
Wen Lu ◽  
Michael Winding ◽  
Margot Lakonishok ◽  
Jill Wildonger ◽  
Vladimir I. Gelfand
Keyword(s):  
Cytoplasmic Streaming ◽  
Cell Types ◽  
Nurse Cells ◽  
Wild Type ◽  
Actin Cortex ◽  
Microtubule Motor ◽  
Multiple Cell ◽  
Cytoplasmic Microtubules ◽  
Two Populations

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

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