scholarly journals N-Glycans of ADAMTS13 modulate its secretion and von Willebrand factor cleaving activity

Blood ◽  
2009 ◽  
Vol 113 (4) ◽  
pp. 929-935 ◽  
Author(s):  
Wenhua Zhou ◽  
Han-Mou Tsai
Keyword(s):  
Von Willebrand Factor ◽  
Golgi Complex ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Glycosylation Sites ◽  
Severe Deficiency ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Severe deficiency of ADAMTS13, a plasma metalloprotease, leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains 10 putative N-glycosylation sites in or near its metalloprotease sequence, spacer region, thrombospondin type 1 repeat no. 4 (TSR no. 4), and CUB domains. Tunicamycin treatment markedly decreased the secretion of ADAMTS13 into the culture medium of transfected cells. Nevertheless, the protease was efficiently secreted from N-acetylglucosaminyltransferase I–deficient Lec1 Chinese hamster ovary cells, indicating that N-glycosylation in the endoplasmic reticulum, but not the conversion of oligomannose to complex N-glycans in the Golgi complex, is important for secretion. However, ADAMTS13 with oligomannose N-glycans cleaved its substrate, von Willebrand factor (VWF) multimers, less effectively, with a higher Km but similar kcat value. In mutagenesis analysis, decreased secretion and VWF cleaving activity was observed with the N146Q and N828Q mutants, while decreased secretion only was observed with the N552Q mutant of ADAMTS13. Enzymatic removal of N-glycans from ADAMTS13 did not affect its VWF cleaving activity. Thus, N-glycosylation is necessary for efficient secretion of ADAMTS13, while conversion of the N-glycans from oligomannose to complex type in the Golgi complex enhances the proteolytic activity of the protease toward VWF multimers. After its secretion, ADAMTS13 does not require N-glycans for its VWF cleaving activity.

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.

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.

Removing the Covalent Links between GP Ibα and GP Ibβ Hampers Binding of von Willebrand Factor to GP Ib-IX Complex in CHO Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3641-3641
Author(s):  
Xi Mo ◽  
Shi-Zhong Luo ◽  
Yuandong Peng ◽  
Adam D. Munday ◽  
Carol Sun ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Cell Model ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Binding Activity ◽  
Ovary Cells ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In the glycoprotein (GP) Ib-IX complex, GP Ibα contains the binding region for its ligand von Willebrand factor (vWF). How the other subunits in the complex, GP Ibβ and GP IX, regulate the GP Ibα-vWF interaction is not clear. Since GP Ibα connects with two GP Ibβ subunits via disulfide bonds, these inter-subunit covalent links may be important to the proper vWF-binding activity of the GP Ib-IX complex. To test this idea, we have obtained Chinese hamster ovary cells stably expressing the mutant GP Ib-IX complex (CHOαSSβIX) in which both residues C484 and C485 of GP Ibα were changed to Ser. As expected, the mutant GP Ibα did not form any disulfide bonds with GP Ibβ in these cells. Nonetheless, co-immunoprecipitation experiments in the digitonin-containing buffer demonstrated that association of GP Ibα with GP Ibβ and GP IX was retained in CHOαSSβIX cells. The expression level of GP Ibα in CHOαSSβIX cells, detected by its binding to WM23, a conformation-insensitive antibody, was comparable to that in the transfected cells stably expressing the wild type GP Ib-IX complex (CHOαβIX). In contrast, binding of CHOαSSβIX cells to AK2, SZ2 or AN51, all of which are GP Ibα-specific conformation-sensitive antibodies, was significantly different from that of CHOαβIX cells. Compared to CHOαβIX cells, ristocetin-induced binding of CHOαSSβIX cells to vWF under static conditions was reduced by about 50% as determined by flow cytometry. Consistently, rolling of CHOαSSβIX cells on the vWF-coated glass slide was significantly faster than CHOαβIX cells under various flow conditions. Thus, in a transfected cell model, removing the disulfide links between GP Ibα and GP Ibβ in the GP Ib-IX complex leads to an alteration in the conformation of the ligand-binding domain in GP Ibα and as a consequence hampers the complex binding to vWF. Our finding suggests that the relative position of GP Ibα and GP Ibβ in the receptor complex is crucial to its vWF-binding activity, which may be exploited to modulate the GP Ibα-vWF interaction.

ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 4033-4039 ◽  
Author(s):  
Jing-fei Dong ◽  
Joel L. Moake ◽  
Leticia Nolasco ◽  
Aubrey Bernardo ◽  
Wendy Arceneaux ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Shear Stresses ◽  
Prolonged Incubation ◽  
Ovary Cells ◽  
Endothelial Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Thrombotic thrombocytopenic purpura (TTP) is a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). The disorder is associated with a deficiency of the VWF-cleaving metalloprotease, ADAMTS-13, with consequent accumulation of ultralarge (UL) VWF multimers in the plasma. ULVWF multimers, unlike plasma forms of VWF, attach spontaneously to platelet GP Ibα, a component of the GP Ib-IX-V complex. We have found that ULVWF multimers secreted from stimulated endothelial cells (ECs) remained anchored to the endothelial surface where platelets and Chinese hamster ovary cells expressing the GP Ib-IX-V complex attached to form long beads-on-a-string structures in the presence of fluid shear stresses in both the venous (2.5 dyne/cm2) and arterial (20 and 50 dyne/cm2) ranges. Although measurement of the activity of the ADAMTS-13 VWF-cleaving metalloprotease in vitro requires prolonged incubation of the enzyme with VWF under nonphysiologic conditions, EC-derived ULVWF strings with attached platelets were cleaved within seconds to minutes in the presence of normal plasma (containing approximately 100% ADAMTS-13 activity) or in the presence of partially purified ADAMTS-13. By contrast, the strings persisted for the entire period of perfusion (10 minutes) in the presence of plasma from patients with TTP containing 0% to 10% ADAMTS-13 activity. These results suggest that cleavage of EC-derived ULVWF multimers by ADAMTS-13 is a rapid physiologic process that occurs on endothelial cell surfaces.

The N-Glycans Modulate the Binding of ADAMTS13 with von Willebrand Factor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2710-2710
Author(s):  
Wenhua Zhou ◽  
Han-Mou Tsai
Keyword(s):  
Von Willebrand Factor ◽  
Chinese Hamster ◽  
Complex Type ◽  
Molecular Weights ◽  
Igg Binding ◽  
Severe Deficiency ◽  
Pngase F ◽  
High Mannose ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13, a circulating multidomain protease consisting a metalloprotease (MP), a disintegrin, a thrombospondin type 1 repeat [TSR], a cysteine rich region, a spacer, 7 additional TSR and 2 CUB domains, is critical for maintaining the homeostasis of von Willebrand factor (VWF). Severe deficiency of the enzyme can lead to VWF-platelet thrombosis in the microcirculation characteristic of thrombotic thrombocytopenic purpura (TTP). Sequence analysis predicts that ADAMTS13 contains 2 and 6 likely N-glycosylation sites respectively at or near the MP and spacer domains, and 2 possible sites in the CUB domains. To investigate the N-glycosylation status and the role of N-glycans in the function of ADAMTS13, we digested plasma derived ADAMTS13 and recombinant ADAMTS13 or its truncated variants with peptide-N-glycosidase F (PNGase F) or endoglycosidase H. SDS PAGE analysis showed that digestion with PNGase F, but not with endo H, decreased the molecular weights of plasma derived ADAMTS13 as well as recombinant ADAMTS13 proteins, including AD7 (residues 1-1427, full-length), AD5 (residues 1-745, MP-TSR#1), AD2 (residues 1-448, MP-TSR#1), AD8 (residues 556-748, spacer-TSR#2) and AD13 (residues 1016-1427, TSR#8-CUB#2), indicating that the MP, spacer and CUB domains were all N-glycosylated as predicted, and contained complex type carbohydrate chains. Enzyme linked immunoassay showed that removal of the N-glycans did not affect the binding of proteases to immobilized VWF multimers or VWF73 peptide. Removal of the N-glycans modestly affected the protease-TTP IgG binding, increasing the Kd by 190% for AD7 and by 50% for AD5. Expression experiments in Lec1 mutant Chinese hamster ovary (CHO) cells with defective GlcNAc-TI activity due to mutations in the Mgat1 gene showed that the resulting high-mannose type AD7 and AD5 variant proteases were secreted normally, showed no difference in VWF cleaving and TTP IgG binding activities, but exhibited increased binding to VWF multimers or VWF73 peptide. In summary, ADAMTS13 are N-glycosylated with complex type oligosaccharides at the MP, spacer and CUB domains. Intracellular conversion from high mannose to complex type glycans modulates the binding of the protease to VWF, suggesting that this process may help prevent depletion of the protease under conditions of high VWF levels in the circulation.

Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with shear-dependent interactions

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 162-168 ◽  
Author(s):  
Jing-Fei Dong ◽  
Michael C. Berndt ◽  
Alicia Schade ◽  
Larry V. McIntire ◽  
Robert K. Andrews ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Platelet Glycoprotein ◽  
Extracellular Region ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Under conditions of high shear stress, both hemostasis and thrombosis are initiated by the interaction of the platelet membrane glycoprotein (GP) Ib-IX-V complex with its adhesive ligand, von Willebrand factor (vWF), in the subendothelial matrix or plasma. This interaction involves the A1 domain of vWF and the N-terminal extracellular region of GP Ibα (His-1-Glu-282), and it can also be induced under static conditions by the modulators ristocetin and botrocetin. In this study, a panel of anti-vWF and anti-GP Ibα antibodies—previously characterized for their effects on ristocetin- and botrocetin-dependent vWF–GP Ib-IX-V interactions—was analyzed for their capacity to inhibit either the adhesion of Chinese hamster ovary cells expressing recombinant GP Ibα to surface-associated vWF under hydrodynamic flow or shear-stress–induced platelet aggregation. The combined results suggest that the shear-dependent interactions between vWF and GP Ibα closely correlate with ristocetin- rather than botrocetin-dependent binding under static conditions and that certain anti-vWF monoclonal antibodies are able to selectively inhibit shear-dependent platelet aggregation.

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.

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