The Effect of N-linked Glycosylation on Molecular Weight, Thrombin Cleavage, and Functional Activity of Human Protein S

1997 ◽  
Vol 77 (06) ◽  
pp. 1156-1163 ◽  
Author(s):  
Deshun Lu ◽  
Rong-Lin Xie ◽  
Andrzej Rydzewski ◽  
George L Long
Keyword(s):  
Specific Activity ◽  
Consensus Sequence ◽  
Protein S ◽  
Human Protein ◽  
Expression Vectors ◽  
Lag Phase ◽  
Similar Amount ◽  
Wild Type ◽  
Glycosylation Sites ◽  
Sds Page

SummaryHuman protein S (HPS) has three potential N-linked glycosylation sites at Asn458’468’489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458→Gln, Ser460→Gly; Asn468→Gln, Thr470→Gly; Asn489→Gln, Thr491→Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of ~78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by α-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser460) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.

scholarly journals Lecithin:cholesterol acyltransferase: role of N-linked glycosylation in enzyme function

1993 ◽  
Vol 294 (3) ◽  
pp. 879-884 ◽  
Author(s):  
K O ◽  
J S Hill ◽  
X Wang ◽  
R McLeod ◽  
P H Pritchard
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Fold Increase ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Lecithin:Cholesterol Acyltransferase ◽  
Mutant Proteins ◽  
Glycosylation Sites

Lecithin:cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) is a glycoprotein which is responsible for the formation of cholesteryl ester in plasma. The carbohydrate content has been estimated to be approx. 25% of the total LCAT mass, and four potential N-linked glycosylation sites have been predicted at residues 20, 84, 272 and 384 of the LCAT protein sequence. In the present study, we have examined which of these sites are utilized and how the N-glycosylation affects the secretion and function of the enzyme. Site-directed mutagenesis was performed to eliminate the glycosylation consensus sequence at each of the four potential sites, and the mutant proteins were expressed in COS cells. The amount of each mutant LCAT secreted was similar to that of the wild-type enzyme but the molecular mass was decreased by 3-4 kDa. The specific activity of each mutant LCAT was significantly different from the wild-type; however, the magnitude and direction of the change depended on the glycosylation site mutagenized. Loss of carbohydrate at position 20, 84 or 272 resulted in a decrease in the specific activity of the mutant enzymes by 18%, 82%, and 62% respectively. In contrast, the mutant protein without glycosylation at position 384 displayed a 2-fold increase in enzyme activity. In addition, a quadruple mutant was constructed such that all four potential glycosylation sites were eliminated. The amount of the unglycosylated LCAT secreted into the culture medium was less than 10% of the wild-type level and the specific activity of this enzyme was decreased to 5% of that of the wild type. The results demonstrate that all four potential N-glycosylation sites in LCAT are used and the presence of carbohydrate at each site has diverse effects on the enzyme activity.

scholarly journals Protein S is Protective in Acute Lung Injury by Inhibiting Cell Apoptosis

2019 ◽  
Vol 20 (5) ◽  
pp. 1082 ◽  
Author(s):  
Prince Baffour Tonto ◽  
Taro Yasuma ◽  
Tetsu Kobayashi ◽  
Corina D’Alessandro-Gabazza ◽  
Masaaki Toda ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Transgenic Mice ◽  
Lung Tissue ◽  
Cell Apoptosis ◽  
Inflammatory Cell ◽  
Protein S ◽  
Inflammatory Cell Infiltration ◽  
Human Protein ◽  
Wild Type

Acute lung injury is a fatal disease characterized by inflammatory cell infiltration, alveolar-capillary barrier disruption, protein-rich edema, and impairment of gas exchange. Protein S is a vitamin K-dependent glycoprotein that exerts anticoagulant, immunomodulatory, anti-inflammatory, anti-apoptotic, and neuroprotective effects. The aim of this study was to evaluate whether human protein S inhibits cell apoptosis in acute lung injury. Acute lung injury in human protein S transgenic and wild-type mice was induced by intratracheal instillation of lipopolysaccharide. The effect of human protein S on apoptosis of lung tissue cells was evaluated by Western blotting. Inflammatory cell infiltration, alveolar wall thickening, myeloperoxidase activity, and the expression of inflammatory cytokines were reduced in human protein S transgenic mice compared to the wild-type mice after lipopolysaccharide instillation. Apoptotic cells and caspase-3 activity were reduced while phosphorylation of extracellular signal-regulated kinase was enhanced in the lung tissue from human protein S transgenic mice compared to wild-type mice after lipopolysaccharide instillation. The results of this study suggest that human protein S is protective in lipopolysaccharide-induced acute lung injury by inhibiting apoptosis of lung cells.

Monoclonal Antibodies Directed to the Calcium-Free Conformation of Human Protein S

1989 ◽  
Vol 62 (02) ◽  
pp. 708-714 ◽  
Author(s):  
Santica Marcovina ◽  
Raffaella Coppola ◽  
Carla Valsecchi ◽  
Adele Zoppo ◽  
Cecilia Gelfi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Protein ◽  
Antigenic Site ◽  
Fluid Phase ◽  
Protein S ◽  
Human Protein ◽  
Sephadex Column ◽  
Sds Page ◽  
Igg1 Subclass ◽  
Sepharose 4B

SummaryFour mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/1 CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate absorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/1 CaCl2. These studies demonstrate that EDTA-dependent monoclonal antibodies can simplify the isolation of PS from human plasma.

Construction and Characterization of Thrombin-resistant Variants of Recombinant Human Protein S

1994 ◽  
Vol 72 (05) ◽  
pp. 693-697 ◽  
Author(s):  
Glenn T G Chang ◽  
Leonie Aaldering ◽  
Tilman M Hackeng ◽  
Pieter H Reitsma ◽  
Rogier M Bertina ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Protein S ◽  
Coagulation Factors ◽  
Human Protein ◽  
Wild Type ◽  
Arginine Residues ◽  
Cofactor Activity

SummaryProtein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and Villa.Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment.All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity. Finally, all variants bind to C4b-binding protein with an affinity similar as the wild type recombinant molecule.

Proteolytic Processing of Big Endothelin-3 by the Kell Blood Group Protein

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1440-1450 ◽  
Author(s):  
Soohee Lee ◽  
Melissa Lin ◽  
Aldo Mele ◽  
Ying Cao ◽  
James Farmar ◽  
...  
Keyword(s):  
Blood Group ◽  
Consensus Sequence ◽  
Large Family ◽  
Protein S ◽  
Negative Control ◽  
Proteolytic Processing ◽  
Wild Type ◽  
Ph Optimum ◽  
Group Protein ◽  
Endothelin 3

Abstract Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp21-Ile22, yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp21-Val22, yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.

scholarly journals Purification of macrophage deactivating factor.

1990 ◽  
Vol 171 (4) ◽  
pp. 1347-1361 ◽  
Author(s):  
S Srimal ◽  
C Nathan
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fluid Phase ◽  
Protein S ◽  
Peritoneal Macrophages ◽  
Reversed Phase ◽  
Tgf Beta ◽  
Sds Page

Macrophage deactivation factor (MDF) in P815 tumor cell-conditioned medium was assayed by its suppression of the ability of activated mouse peritoneal macrophages to release hydrogen peroxide. MDF displayed properties of a soluble protein(s) associated with both low (8-25,000) and high (greater than 450,000) Mr fractions. MDF was purified 6,140-fold by a seven-step procedure: extraction with acid-ethanol; precipitation with ether; and fractionation on gel filtration, anion-exchange, diphenyl reversed-phase and C4 reversed-phase HPLC columns, the last column twice. The final preparation contained two species: (a) a approximately 13,000 Mr band on reducing or nonreducing SDS-PAGE and on autoradiograms after radioiodination with chloramine T, and (b) a 66,000 Mr species ranging from approximately 5% to approximately 50% of the protein detectable by silver strain. The 66,000 Mr species was identified as albumin from its NH2-terminal amino acid sequence. However, no amino acid sequence could be obtained for the approximately 13,000 Mr species, either in fluid phase or after electroelution of the corresponding SDS-PAGE band. Thus, approximately 13,000 Mr MDF associates tightly with albumin through a variety of separation techniques, and may have a blocked NH2 terminus. Purified MDF afforded 50% inhibition of activated macrophage H2O2 releasing capacity at a concentration of 1-10 nM. Separation of MDF from most higher Mr moieties was associated with disproportionately small increases in specific activity, suggesting MDF might be partially inactivated by purification. As purified, MDF was approximately 1,000-fold less potent at deactivating macrophages than TGF-beta. Antibodies that neutralized the macrophage-deactivating effect of TGF-beta did not inhibit deactivation by MDF.

CHARACTERIZATION OF SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR MUTANTS PRODUCED BY SITE-SPECIFIC MUTAGENESIS OF LYS

1987 ◽  
Author(s):  
L Nelles ◽  
H R Linjnen ◽  
E Demarsin ◽  
D Collen ◽  
W E Holmes
Keyword(s):  
Plasminogen Activator ◽  
Specific Activity ◽  
Chinese Hamster ◽  
Expression Vectors ◽  
Wild Type ◽  
Single Chain ◽  
Plasma Clot ◽  
Serum Free ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

A cDNA encoding full length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced. The recombinant scu-PA (rscu-PA) cDNA as well as the cDNA of two mutants constructed by deoxyoligonucleotide directed mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158 ) or to Glu158 (rscu-PA-Glu158 ) were inserted into SV40 early promoter/enhancer based expression vectors, which were used to transfect Chinese Hamster Ovary (CHO) cells. The expression products were purified from serum-free conditioned media by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration.The amidolytic activity of the three rscu-PAs was low (< 500 IU/mg). The mutant rscu-PAs, in contrast to the rscu-PA and nscu-PA, could not be converted into an amidolytically active two-chain form (tcu-PA) by plasmin. The mutant scu-PAs had a very low specific activity (< 1,000 IU/mg) on fibrin plates, whereas wild type rscu-PA had a specific activity < 1000 IU/mg. The mutant scu-PAs did not cause lysis of a I-fibrin labeled plasma clot immersed in citrated human plasma. Serum-free medium from a control transfected CHO cell line showed no significant plasminogen activating activity.In a purified system, both rscu-PA-Gly and rscu-PA-Glu activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (K = 60-80 yM) but with a higher catalytic rate constant (k2 = B.01 s-1) as compared to the wild type rscu-PA (K =1.0 yM, k = 0.002 s-1).It is concluded thaz conversion of scu-PA to tcu-PA is prerequisite for the activation of plasminogen. However, Lys158 seems to be important for the stability of the Michaelis complex between scu-PA and plasminogen.

Studies of the Interaction between Human Protein S and Human C4b-Binding Protein Using Deletion Variants of Recombinant Human Protein S

1994 ◽  
Vol 71 (04) ◽  
pp. 461-467 ◽  
Author(s):  
Glenn T G Chang ◽  
Bart H A Maas ◽  
Hans K Ploos van Amstel ◽  
Pieter H Reitsma ◽  
Rogier M Bertina ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Recombinant Proteins ◽  
Protein S ◽  
Human Protein ◽  
Amino Acid Residues ◽  
Wild Type ◽  
The Third ◽  
Terminal Loop ◽  
Cofactor Activity ◽  
Recombinant Human Protein

SummaryHuman protein S interacts noncovalently with human C4b-binding protein (C4BP). We have studied this interaction using deletion variants of recombinant human protein S. Two deletion variants were constructed by restriction enzyme digestion and in vitro site-specific mutagenesis of the human protein S cDNA. The variants were stably expressed in Cl27 cells. Recombinant proteins were purified using Fast Flow Q anion-exchange chromatography. The activated protein C (APC) cofactor activity, C4BP binding properties and reactivity to different monoclonal antibodies against human protein S were examined. The first variant (E variant), which has a deletion of the third epidermal growth factor (EGF)-like domain (deletion of exon VII, corresponding to amino acid residues ASP-160 to Asp-202) expresses normal APC cofactor • activity in a plasma system. This activity was inhibited by the addition of purified C4BP. The second variant (L variant), which has a deletion of the C-terminal loop of the sex hormone binding globulin (SHBG)- like domain (deletion of exon XV, corresponding to amino acid residues Asp-583 to Ser-635) also expresses normal APC cofactor activity in plasma. This activity could only be partially inhibited by the addition of purified C4BP.Binding of the recombinant proteins to C4BP was studied in a system using purified proteins. The E variant binds to C4BP with the same affinity similar as recombinant wild type protein S (apparent Kd ∼ 10−10 M). The L variant, however, shows a markedly reduced affinity for binding to C4BP (apparent Kd ∼ 10−7 M).Three different Ca2+-independent monoclonal antibodies (S5, S12, S17) against protein S, which do not interfere with the APC cofactor activity and C4BP binding of protein S, were used to screen the deletion variants for possible conformational changes. Two of these showed the same affinity for the E and L variant as for wild type recombinant protein S. The third, S12, which recognizes an epitope in the vicinity of ser-460, reacts normally with the E variant but has a strongly reduced affinity for the L variant, although the presence of the epitope could be clearly demonstrated by immunoblotting under denaturing conditions.This suggests that the deletion of the C-terminal loop induces a conformational change in protein S which affects the epitope for S12. Therefore although our results indicate that the C-terminal loop of the SHBG-like domain of human protein S is involved in the interaction with C4BP, we cannot exclude the possibility that the deletion of the C-terminal loop induces a conformational change that results in a loss of binding affinity for C4BP elsewhere in the protein S molecule.

Proteolytic Processing of Big Endothelin-3 by the Kell Blood Group Protein

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1440-1450 ◽  
Author(s):  
Soohee Lee ◽  
Melissa Lin ◽  
Aldo Mele ◽  
Ying Cao ◽  
James Farmar ◽  
...  
Keyword(s):  
Blood Group ◽  
Consensus Sequence ◽  
Large Family ◽  
Protein S ◽  
Negative Control ◽  
Proteolytic Processing ◽  
Wild Type ◽  
Ph Optimum ◽  
Group Protein ◽  
Endothelin 3

Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp21-Ile22, yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp21-Val22, yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
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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