scholarly journals A mammalian fatty acid hydroxylase responsible for the formation of α-hydroxylated galactosylceramide in myelin

2005 ◽  
Vol 388 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Matthias ECKHARDT ◽  
Afshin YAGHOOTFAM ◽  
Simon N. FEWOU ◽  
Inge ZÖLLER ◽  
Volkmar GIESELMANN
Keyword(s):  
Fatty Acid ◽  
Functional Role ◽  
Time Course ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mammalian Brain ◽  
Fatty Acid Hydroxylase

Hydroxylation is an abundant modification of the ceramides in brain, skin, intestinal tract and kidney. Hydroxylation occurs at the sphingosine base at C-4 or within the amide-linked fatty acid. In myelin, hydroxylation of ceramide is exclusively found at the α-C atom of the fatty acid moiety. α-Hydroxylated cerebrosides are the most abundant lipids in the myelin sheath. The functional role of this modification, however, is not known. On the basis of sequence similarity to a yeast C26 fatty acid hydroxylase, we have identified a murine cDNA encoding FA2H (fatty acid 2-hydroxylase). Transfection of FA2H cDNA in CHO cells (Chinese-hamster ovary cells) led to the formation of α-hydroxylated fatty acid containing hexosylceramide. An EGFP (enhanced green fluorescent protein)–FA2H fusion protein co-localized with calnexin, indicating that the enzyme resides in the endoplasmic reticulum. FA2H is expressed in brain, stomach, skin, kidney and testis, i.e. in tissues known to synthesize fatty acid α-hydroxylated sphingolipids. The time course of its expression in brain closely follows the expression of myelin-specific genes, reaching a maximum at 2–3 weeks of age. This is in agreement with the reported time course of fatty acid α-hydroxylase activity in the developing brain. In situ hybridization of brain sections showed expression of FA2H in the white matter. Our results thus strongly suggest that FA2H is the enzyme responsible for the formation of α-hydroxylated ceramide in oligodendrocytes of the mammalian brain. Its further characterization will provide insight into the functional role of α-hydroxylation modification in myelin, skin and other organs.

Role of the α-subunit326GRV sequence in the surface expression of fibrinogen and vitronectin receptors

1998 ◽  
Vol 275 (5) ◽  
pp. C1239-C1246 ◽  
Author(s):  
Milagros Ferrer ◽  
Matilde S. Ayuso ◽  
Nora Butta ◽  
Roberto Parrilla ◽  
Consuelo González-Manchón
Keyword(s):  
Functional Role ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Platelet Surface ◽  
Α Subunit ◽  
Vitronectin Receptor ◽  
Ovary Cells ◽  
High Affinity

The platelet GPIIb-GPIIIa heterodimer (integrin αIIbβ3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The326GRV motif in GPIIb is highly conserved in the α-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327→His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327in the α-subunit of the vitronectin receptor, to His did not prevent the surface expression of αv-GPIIIa complexes. Thus the conformational constraint imposed by His327seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.

scholarly journals Cytoplasmic domains of the reduced folate carrier are essential for trafficking, but not function

2002 ◽  
Vol 364 (3) ◽  
pp. 777-786 ◽  
Author(s):  
Heather SADLISH ◽  
Frederick M.R. WILLIAMS ◽  
Wayne F. FLINTOFF
Keyword(s):  
Protein Function ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Reduced Folate Carrier ◽  
Cytoplasmic Domains ◽  
Intracellular Loop ◽  
Cytoplasmic Loop ◽  
Substrate Transport

The reduced folate carrier (RFC) protein has a secondary structure consistent with the predicted 12 transmembrane (TM) domains, intracellular N- and C-termini and a large cytoplasmic loop between TM6 and TM7. In the present study, the role of the cytoplasmic domains in substrate transport and protein biogenesis were examined using an array of hamster RFC deletion mutants fused to enhanced green fluorescent protein and expressed in Chinese hamster ovary cells. The N- and C-terminal tails were removed both individually and together, or the large cytoplasmic loop was modified such that the domain size and role of conserved sequences could be examined. The loss of the N- or C-terminal tails did not appear to significantly disrupt protein function, although both termini appeared to have a role in the efficiency with which molecules exited the endoplasmic reticulum to localize at the plasma membrane. There appeared to be both size and sequence requirements for the intracellular loop, which are able to drastically affect protein stability and function unless met. Furthermore, there might be an indirect role for the loop in substrate translocation, since even moderate changes significantly reduced the Vmax for methotrexate transport. Although these cytoplasmic domains do not appear to be absolutely essential for substrate transport, each one is important for biogenesis and localization.

scholarly journals Functional Role of Na+/H+ Exchanger in Ca2+ Influx Mediated via Human Endothelin Type A Receptor Stably Expressed in Chinese Hamster Ovary Cells

2008 ◽  
Vol 107 (4) ◽  
pp. 456-459 ◽  
Author(s):  
Takahiro Horinouchi ◽  
Yumie Miyake ◽  
Tadashi Nishiya ◽  
Arata Nishimoto ◽  
Shigeru Morishima ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Functional Role ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Type A ◽  
Ovary Cells ◽  
Endothelin Type A Receptor

scholarly journals Peptide Antagonists That Inhibit Sin Nombre Virus and Hantaan Virus Entry through the β3-Integrin Receptor

2005 ◽  
Vol 79 (12) ◽  
pp. 7319-7326 ◽  
Author(s):  
Richard S. Larson ◽  
David C. Brown ◽  
Chunyan Ye ◽  
Brian Hjelle
Keyword(s):  
Viral Entry ◽  
Sequence Similarity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Phage Display Library ◽  
Hantaan Virus ◽  
Sin Nombre Virus ◽  
Fab Fragment ◽  
Ovary Cells

ABSTRACT Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the αvβ3 integrin, and transfection of human β3 integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-β3 antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified αvβ3, we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 μg/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use β3 integrins and PHV uses a different receptor, β1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via β3 integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.

Abstract 11947: Unraveling the Mechanism for the Stimulatory Role of Platelet GPIb-IX-V in Thrombin-Induced Platelet Activation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Brian Estevez ◽  
Michael K Delaney ◽  
Aleksandra Stojanovic-Terpo ◽  
Xiaoping Du
Keyword(s):  
Platelet Activation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Inhibitory Peptide ◽  
Protease Activated Receptors ◽  
Signaling Mechanism ◽  
Ovary Cells

Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. Pretreatment of human platelets with MPalphaC, an inhibitory peptide based on a critical 14-3-3 signaling protein binding site on the cytoplasmic domain of the GPIb alpha chain, inhibited thrombin-induced platelet activation. MPalphaC-treatment inhibited thrombin-induced activation of Rac1 and LIMK1, both of which are known to play essential roles in GPIb signaling. To more specifically determine the role of GPIb-IX, we reconstituted GPIb-IX-facilitated thrombin signaling in Chinese Hamster Ovary cells expressing PAR1. Thrombin induced signaling was significantly enhanced by GPIb-expression, and deletion of the cytoplasmic 14-3-3-binding domain of GPIb alpha abolished the stimulatory effect of GPIb on thrombin signaling. Furthermore, the role of GPIb-IX in promoting thrombin signaling requires Rac1, and GPIb-IX-dependent Rac1 activation and LIMK phosphorylation are abolished in delta 605 cells expressing a 14-3-3-binding defective mutant GPIb alpha. Taken together, these data suggest that the stimulatory role of GPIb in thrombin signaling requires a C-terminal 14-3-3-binding region which mediates activation of a Rac1/LIMK1 pathway that promotes thrombin signaling leading to platelet activation.

scholarly journals Hydrophobic Regions Adjacent to Transmembrane Domains 1 and 5 Are Important for the Targeting of the 70-kDa Peroxisomal Membrane Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33831-33844 ◽  
Author(s):  
Yoshinori Kashiwayama ◽  
Kota Asahina ◽  
Masashi Morita ◽  
Tsuneo Imanaka
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH2-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH2-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile307/Leu308) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4063-4070
Author(s):  
A J Dorner ◽  
M G Krane ◽  
R J Kaufman
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary ◽  
Heterologous Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Ovary Cells ◽  
Tissue Plasminogen ◽  
Stable Association

GRP78 is localized in the endoplasmic reticulum and associates with improperly folded or underglycosylated proteins. The role of GRP78 in secretion was studied in Chinese hamster ovary cells expressing a tissue plasminogen activator (tPA) variant which lacks potential N-linked glycosylation site sequences because of mutagenesis. The expression of variant tPA resulted in elevated levels of GRP78 and its stable association with tPA. The introduction of antisense GRP78 genes resulted in a two- to threefold reduction in GRP78 levels compared with those of the original cells. Cells with reduced levels of GRP78 secreted two- to threefold-higher levels of tPA activity. tPA expressed in these cells displayed reduced association with GRP78, and a greater proportion was processed to the mature form and secreted. These results demonstrate that reduction of GRP78 level can improve the secretion of an associated protein.

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