Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX

2001 ◽  
Vol 358 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Philippe ULSEMER ◽  
Catherine STRASSEL ◽  
Marie-Jeanne BAAS ◽  
Jean SALAMERO ◽  
Sylvette CHASSEROT-GOLAZ ◽  
...  
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Ovary Cells ◽  
Confocal Immunofluorescence Microscopy ◽  
Vessel Injury ◽  
Von Willebrand

The multisubunit leucine-rich glycoprotein (GP) Ib–IX–V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard–Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse–chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbα undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbα and surface expression of the complex. Biosynthetic studies were then performed on a Bernard–Soulier variant based on previous description of abnormal GPIbα size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbα. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments (‘ERGIC’) and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

Quaternary Organization of GPIb-IX Complex and Insights Into Bernard-Soulier Syndrome Revealed by the Crystal Structures of GPIbβ Ectodomain and a GPIbβ/GPIX Chimera

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2195-2195
Author(s):  
Paul A. McEwan ◽  
Wenjun Yang ◽  
Katherine H. Carr ◽  
Xi Mo ◽  
Xiaofeng Zheng ◽  
...  
Keyword(s):  
Structural Model ◽  
Conflicts Of Interest ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Surface ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Ovary Cells ◽  
Bernard Soulier Syndrome

Abstract Abstract 2195 Platelet glycoprotein (GP)Ib-IX receptor complex contains three subunits, GPIbα, GPIbβ and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome (BSS). We have crystallized the GPIbβ ectodomain (GPIbβE) and determined the structure to reveal a single leucine-rich repeat with N- and C-terminal disulfide bonded capping regions. The central region of the structure can be divided into concave parallel β-sheet and convex loops. The crystal structure of a GPIbβE/GPIXE chimera that contains three non-continguous convex loops of GPIX and retains a GPIbβ-binding site of GPIX (Mo et al. J. Thromb. Haemost. 7:1533–40, 2009) was also determined. The chimera, but not GPIbβE, forms a homotetramer in the crystal, revealing a quaternary interface between GPIbβ and GPIX ectodomains. Central to this interface is residue Tyr106 from GPIbβ that inserts into a shallow and largely hydrophobic pocket generated by two convex loops from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIbβ and GPIX in the full-length complex. Eight GPIbβ missense mutations identified from BSS patients were examined in transiently transfected Chinese hamster ovary cells for changes to the GPIb-IX complex surface expression. Six of the eight mutations lead to secretion defect and/or misfolding of GPIbβE. In contrast, the other two mutations, A108P and P74R, were found to maintain normal secretion and folding of GPIbβE but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIbβE interface with GPIX indicates that residues Ala108 and Pro74 in GPIbβ are located at the GPIbβE/GPIXE interfaces. Based on the tetrameric arrangement of the chimera structure, we propose a structural model for the GPIb-IX complex that embodies its organizing principles and helps to provide mechanistic insights on its assembly, function and regulation. Disclosures: No relevant conflicts of interest to declare.

Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ibα using canine–human chimeras

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 145-150 ◽  
Author(s):  
Yang Shen ◽  
Jing-fei Dong ◽  
Gabriel M. Romo ◽  
Wendy Arceneaux ◽  
Andrea Aprico ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Platelet Glycoprotein ◽  
Flow Conditions ◽  
Flanking Sequence ◽  
Ovary Cells ◽  
Leucine Rich Repeats ◽  
Von Willebrand ◽  
Willebrand Factor

Platelet glycoprotein Ib-IX-V (GPIb-IX-V) mediates adhesion to von Willebrand factor (vWF) in (patho)physiological thrombus formation. vWF binds the N-terminal 282 residues of GPIbα, consisting of an N-terminal flank (His1–Ile35), 7 leucine-rich repeats (Leu36–Ala200), a C-terminal flank (Phe201–Gly268), and a sulfated tyrosine sequence (Asp269–Glu282). By expressing canine–human chimeras of GPIbα on Chinese hamster ovary cells, binding sites for functional anti-GPIbα antibodies to individual domains were previously mapped, and it was shown that leucine-rich repeats 2 to 4 were required for optimal vWF recognition under static or flow conditions. Using novel canine–human chimeras dissecting the C-terminal flank, it is now demonstrated that (1) Phe201-Glu225 contains the epitope for AP1, an anti-GPIbα monoclonal antibody that inhibits both ristocetin- and botrocetin-dependent vWF binding; (2) VM16d, an antibody that preferentially inhibits botrocetin-dependent vWF binding, recognizes the sequence Val226-Gly268, surrounding Cys248, which forms a disulfide-bond with Cys209; (3) vWF binding to chimeric GPIbα is comparable to wild-type in 2 chimeras in which the sixth leucine-rich repeat was of the same species as the first disulfide loop (Phe201-Cys248) of the C-terminal flank, suggesting an interaction between these domains may be important for optimal vWF binding; and (4) replacing the C-terminal flank second disulfide loop (Asp249-Gly268) in human GPIbα with the corresponding canine sequence enhanced vWF binding under static and flow conditions, providing the first evidence for a gain-of-function phenotype associated with the second loop of the C-terminal flank.

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 A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 135-150 ◽  
Author(s):  
N T Ktistakis ◽  
C Y Kao ◽  
R H Wang ◽  
M G Roth
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Secretory Activity ◽  
Specific Marker ◽  
Ovary Cells ◽  
Fluorescent Lipid

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

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 Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells.

1989 ◽  
Vol 9 (3) ◽  
pp. 1233-1242 ◽  
Author(s):  
R J Kaufman ◽  
L C Wasley ◽  
M V Davies ◽  
R J Wise ◽  
D I Israel ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Translation Product ◽  
Factor Viii Activity ◽  
Ovary Cells ◽  
Von Willebrand ◽  
Willebrand Factor

In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.

Sign in / Sign up

Export Citation Format

Share Document