ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non–heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.

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NEUTRALIZATION OF HEPARIN-LIKE SACCHARIDES BY COMPLEMENT S PROTEIN/VITRONECTIN

10.1055/s-0038-1643636 ◽

1987 ◽

Author(s):

D A Lane ◽

A M Flynn ◽

G Pejler ◽

U Lindahl ◽

K Preissner

Keyword(s):

Heparan Sulfate ◽

Anticoagulant Activity ◽

Protein A ◽

Membrane Attack Complex ◽

Factor Xa ◽

Molar Ratio ◽

Platelet Factor ◽

S Protein ◽

Heparin Activity ◽

Heparin Fractions

S protein, a major inhibitor of the assembly of the membrane attack complex of complement, has recently been shown to be identical to the serum spreading factor vitronectin. It has also been demonstrated to have anti-heparin properties. We have studied the heparin neutralizing properties of S protein/vitronectin using heparin, heparan sulfate and heparin oligosaccharides with high affinity for antithrombin. The ability of heparin fractions, Mr 7300-18800, and of the International Heparin Standard, to accelerate the inactivation of thrombin and Factor Xa by anti thrombin was readily neutralized by S protein/vitronectin. Addition of the protein to the various saccharide fractions at a molar ratio 1-3/1 produced 50/ neutralization, while complete neutralization was achieved at a molar ratio of <10/1. Moreover, S protein/vitronectin efficiently neutralized oligosaccharides of Mr 2400-7200, unlike the two other physiologically occur ing heparin neutralizing proteins histidine-rich glycoprotein <HRG) and platelet factor 4 < PF4) <Lane et al (1986) J. Biol. Chem.261, 3980; Lane et al (1984) Biochem. J. 218, 725). Like PF4, but unlike HRG, S protein/vitronectin readily neutralized the anticoagulant activities of heparan sulfate of Mr ˜20000. These findings indicate that S protein/vitronectin requires little more than the antithrombin-binding pentasaccharide with which to interact in order to express its anti-heparin activity. Furthermore, the results suggest that S protein/vitronectin may be a physiological1y important modulator of the anticoagulant activity of heparin-like material on or near the vascular endothelium.

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Anticoagulant Activity Of Heparan Sulfate From Cerebral Microvascular Tissue

10.1055/s-0038-1652301 ◽

1981 ◽

Author(s):

J A Marcum ◽

L Fritze ◽

R D Rosenberg

Keyword(s):

Heparan Sulfate ◽

Biological Activities ◽

Anticoagulant Activity ◽

Factor Xa ◽

Electrophoretic Analysis ◽

Factor X ◽

Vascular Elements ◽

Specific Species ◽

Two Peaks ◽

Second Peak

Microvascular tissue (MT) was isolated by standard sieving techniques from the cerebral cortex of yearling calves. After proteolysis of MT, the cell debris was pelleted and the supernatant was charged to DEAE-Sepharose A-25 equilibrated with 0.1 M NaCl in 0.01 M Tris-HCl, pH 7.5. Uronic acid determination demonstrated that two peaks of mucopolysaccharides (MS) were eluted at an added salt concentration of 0.4 M NaCl and 0.8 M NaCl, respectively. Enzymatic and electrophoretic analyses of the first peak revealed the presence of a minimally sulfated MS with no ability to accelerate thrombin or factor Xa neutralization by antithrombin (AT). MS isolated from the second peak were degraded by chondroitin ABC lyase and Flavobacterium heparinase. This material migrated slightly behind NIH standard heparan sulfate (HS) and distinctly separately from NIH standard heparin (Hp) which had much greater electrophoretic mobility. Sequential degradation with chondroitinase and heparinase revealed that HS was responsible for the anticoagulant activity of the second peak. The antithrombin and anti-factor X activities of HS were 2.81 u/mg and 2.86 u/mg, respectively. Affinity fractionation of HS with AT resulted in an increase of antithrombin and anti-factor X activities to 36.6 u/mg (13 fold augmentation) and 41.3 u/mg (15 fold augmentation), respectively. Each of the biological activities required the presence of AT and disappeared upon exposure to heparinase. Electrophoretic analysis of the affinity fractionated material demonstrated that it migrated slightly behind NIH standard HS and 40% slower than NIH standard Hp. In conclusion, the ionic strength at which this affinity fractionated HS had been eluted from DEAE-Sepharose and its mobility during electrophoresis clearly distinguish it from classical Hp. These data demonstrate the existence of a specific species of HS from MT with significant anticoagulant activity. These components may in part, be responsible for the non- thrombogenic properties of vascular elements.

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Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646437 ◽

1991 ◽

Vol 66 (04) ◽

pp. 453-458 ◽

Cited By ~ 15

Author(s):

John T Brandt

Keyword(s):

Specific Activity ◽

Anticoagulant Activity ◽

Factor Xa ◽

Clinical Importance ◽

Potential Method ◽

Quantitative Expression ◽

Increased Risk ◽

Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

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The Anticoagulant Properties of a Modified Form of Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647048 ◽

1988 ◽

Vol 60 (02) ◽

pp. 298-304 ◽

Cited By ~ 17

Author(s):

C A Mitchell ◽

S M Kelemen ◽

H H Salem

Keyword(s):

Monoclonal Antibody ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Factor Xa ◽

Protein S ◽

Human Neutrophil Elastase ◽

Factor X ◽

Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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Comparison of the Non-Specific Binding of Unfractionated Heparin and Low Molecular Weight Heparin (Enoxaparin) to Plasma Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649639 ◽

1993 ◽

Vol 70 (04) ◽

pp. 625-630 ◽

Cited By ~ 71

Author(s):

Edward Young ◽

Benilde Cosmi ◽

Jeffrey Weitz ◽

Jack Hirsh

Keyword(s):

Molecular Weight ◽

Unfractionated Heparin ◽

Plasma Proteins ◽

Low Molecular Weight Heparin ◽

Specific Binding ◽

Anticoagulant Activity ◽

Factor Xa ◽

Low Molecular Weight ◽

Heparin Binding ◽

Fixed Amount

SummaryThe non-specific binding of anticoagulantly-active heparin to plasma proteins may influence its anticoagulant effect. We used low affinity heparin (LAH) essentially devoid of anti-factor Xa activity to investigate the extent and possible mechanism of this non-specific binding. The addition of excess LAH to platelet-poor plasma containing a fixed amount of unfractionated heparin doubled the anti-factor Xa activity presumably because it displaces anticoagulantly-active heparin from plasma proteins. Although dextran sulfates of varying molecular weights also increased the anti-factor Xa activity, less sulfated heparin-like polysaccharides had no effect. These findings suggest that the ability to displace active heparin from plasma protein binding sites is related to charge and may be independent of molecular size. In contrast to its effect in plasma containing unfractionated heparin, there was little augmentation in anti-factor Xa activity when LAH was added to plasma containing low molecular weight heparin (LMWH), indicating that LMWH binds less to plasma proteins than unfractionated heparin. This concept is supported by studies comparing the anticoagulant activity of unfractionated heparin and LMWH in plasma with that in buffer containing antithrombin III. The anti-factor Xa activity of unfractionated heparin was 2-fold less in plasma than in the purified system. In contrast, LMWH had identical anti-factor Xa activity in both plasma and buffer, respectively. These findings may be clinically relevant because the recovered anti-factor Xa activity of unfractionated heparin was 33% lower in plasma from patients with suspected venous thrombosis than in plasma from healthy volunteers. The reduced heparin recovery in patient plasma reflects increased heparin binding to plasma proteins because the addition of LAH augmented the anti-factor Xa activity. In contrast to unfractionated heparin, there was complete recovery of LMWH added to patient plasma and little increase of anti-factor Xa activity after the addition of LAH. These findings may explain why LMWH gives a more predictable dose response than unfractionated heparin.

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Anticoagulant Activity of β2-Glycoprotein I Is Potentiated by a Distinct Subgroup of Anticardiolipin Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656368 ◽

1992 ◽

Vol 68 (03) ◽

pp. 297-300 ◽

Cited By ~ 130

Author(s):

Monica Galli ◽

Paul Comfurius ◽

Tiziano Barbui ◽

Robert F A Zwaal ◽

Edouard M Bevers

Keyword(s):

Human Plasma ◽

Anticoagulant Activity ◽

Factor Xa ◽

Type A ◽

Lipid Vesicles ◽

Dependent Manner ◽

Type B ◽

Distinct Subgroup ◽

Glycoprotein I ◽

Thrombin Formation

SummaryPlasmas of 16 patients positive for both IgG anticardiolipin (aCL) antibodies and lupus anticoagulant (LA) antibodies were subjected to adsorption with liposomes containing cardiolipin. In 5 of these plasmas both the anticardiolipin and the anticoagulant activities were co-sedimented with the liposomes in a dose-dependent manner, whereas in the remaining cases only the anticardiolipin activity could be removed by the liposomes, leaving the anticoagulant activity (LA) in the supernatant plasma. aCL antibodies purified from the first 5 plasmas were defined as aCL-type A, while the term aCL-type B was used for antibodies in the other 11 plasmas, from which 2 were selected for this study.Prolongation of the dRVVT was produced by affinity-purified aCL-type A antibodies in plasma of human as well as animal (bovine, rat and goat) origin. aCL-type B antibodies were found to be devoid of anticoagulant activity, while the corresponding supernatants containing LA IgG produced prolongation of the dRVVT only in human plasma.These anticoagulant activities of aCL-type A and of LA IgG's were subsequently evaluated in human plasma depleted of β2-glycoprotein I (β2-GPI), a protein which was previously shown to be essential in the binding of aCL antibodies to anionic phospholipids. Prolongation of the dRVVT by aCL-type A antibodies was abolished using β2-GPI deficient plasma, but could be restored upon addition of β2-GPI. In contrast, LA IgG caused prolongation of the dRVVT irrespective of the presence or absence of β2-GPI.Since β2-GPI binds to negatively-charged phospholipids and impedes the conversion of prothrombin by the factor Xa/Va enzyme complex (Nimpf et al., Biochim Biophys Acta 1986; 884: 142–9), comparison was made of the effect of aCL-type A and aCL-type B antibodies on the rate of thrombin formation in the presence and absence of β2-GPI. This was measured in a system containing highly purified coagulation factors Xa, Va and prothrombin and lipid vesicles composed of 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. No inhibition on the rate of thrombin formation was observed with both types of aCL antibodies when either β2-GPI or the lipid vesicles were omitted. Addition of β2-GPI to the prothrombinase assay in the presence of lipid vesicles causes a time-dependent inhibition which was not affected by the presence of aCL-type B or non-specific IgG. In contrast, the presence of aCL-type A antibodies dramatically increased the anticoagulant effect of β2-GPI. These data indicate that the anticoagulant activity of aCL-type A antibodies in plasma is mediated by β2-GPI.

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DAPLE and MPDZ bind to each other and cooperate to promote apical cell constriction

Molecular Biology of the Cell ◽

10.1091/mbc.e19-02-0091 ◽

2019 ◽

Vol 30 (16) ◽

pp. 1900-1910 ◽

Cited By ~ 6

Author(s):

Arthur Marivin ◽

Mikel Garcia-Marcos

Keyword(s):

G Protein ◽

Cultured Cells ◽

Apical Cell ◽

Cell Junctions ◽

Binding Motif ◽

Apical Constriction ◽

Protein Activation ◽

Neuroepithelial Cells ◽

Two Factors ◽

Apical Junctions

Dishevelled-Associating Protein with a high frequency of LEucines (DAPLE) belongs to a group of unconventional activators of heterotrimeric G-proteins that are cytoplasmic factors rather than membrane proteins of the G-protein–coupled receptor superfamily. During neurulation, DAPLE localizes to apical junctions of neuroepithelial cells and promotes apical cell constriction via G-protein activation. While junctional localization of DAPLE is necessary for this function, the factors it associates with at apical junctions or how they contribute to DAPLE-mediated apical constriction are unknown. MPDZ is a multi-PDZ (PSD95/DLG1/ZO-1) domain scaffold present at apical cell junctions whose mutation in humans is linked to nonsyndromic congenital hydrocephalus (NSCH). DAPLE contains a PDZ-binding motif (PBM) and is also mutated in human NSCH, so we investigated the functional relationship between both proteins. DAPLE colocalized with MPDZ at apical cell junctions and bound directly to the PDZ3 domain of MPDZ via its PBM. Much like DAPLE, MPDZ is induced during neurulation in Xenopus and is required for apical constriction of neuroepithelial cells and subsequent neural plate bending. MPDZ depletion also blunted DAPLE-mediated apical constriction of cultured cells. These results show that DAPLE and MPDZ, two factors genetically linked to NSCH, function as cooperative partners at apical junctions and are required for proper tissue remodeling during early stages of neurodevelopment.

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Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)82186-x ◽

1993 ◽

Vol 268 (14) ◽

pp. 10160-10167

Author(s):

Z.S. Ji ◽

W.J. Brecht ◽

R.D. Miranda ◽

M.M. Hussain ◽

T.L. Innerarity ◽

...

Keyword(s):

Apolipoprotein E ◽

Heparan Sulfate ◽

Cultured Cells ◽

Heparan Sulfate Proteoglycans ◽

Remnant Lipoproteins

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Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

Scientific Reports ◽

10.1038/s41598-021-84551-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alessandro T. Caputo ◽

Oliver M. Eder ◽

Hana Bereznakova ◽

Heleen Pothuis ◽

Albert Ardevol ◽

...

Keyword(s):

Cell Lines ◽

Mammalian Cell ◽

Recombinant Proteins ◽

Cultured Cells ◽

Hek293 Cells ◽

Reaction Products ◽

Enzyme Form ◽

X Ray Crystallography ◽

Mammalian Cell Lines ◽

Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

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