scholarly journals Identification of novel glycosylation events on human serum-derived Factor IX

2019 ◽  
Author(s):  
Cassandra L. Pegg ◽  
Lucia F. Zacchi ◽  
Dinora Roche Recinos ◽  
Christopher B. Howard ◽  
Benjamin L. Schulz
Keyword(s):  
Human Serum ◽  
Serine Protease ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Activate Factor ◽  
Protease Domain ◽  
Structural Characterisation ◽  
Serine Protease Domain ◽  
Human Factor Ix

ABSTRACTHuman Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.

The Activation of Human Factor IX

1975 ◽  
Vol 33 (03) ◽  
pp. 553-563 ◽  
Author(s):  
B Østerud ◽  
K Laake ◽  
H Prydz
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Tissue Thromboplastin ◽  
Native Factor

SummaryThe activation of factor IX purified from human plasma has been studied. Factor XIa and kallikrein separately activated factor IX to factor IXa. In both cases factor IX a had an apparent molecular weight of about 42–45000 in sodium dodecyl sul-phate-polyacrylamide disc gel electrophoresis compared with a molecular weight of about 70000 for the native factor IX. The activation by XIa required Ca2+-ions whereas Ca2+-ions did not influence the activation by kallikrein. A mixture of tissue thromboplastin and factor VII or RusselPs-viper venom alone did not activate factor IX. Trypsin activated and plasmin inactivated factor IX.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

339 BIOENGINEERING OF THE MAMMARY GLAND OF LIVESTOCK: INCREASED PROPEPTIDE PROCESSING OF FACTOR IX IN THE MILK OF TRANSGENIC PIGS BY CO-EXPRESSION OF FURIN

2011 ◽  
Vol 23 (1) ◽  
pp. 265 ◽  
Author(s):  
J. Zhao ◽  
E. Walters ◽  
J. Calcaterra ◽  
J. Ross ◽  
L. Spate ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Production Efficiency ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Blood Proteins ◽  
Wild Type ◽  
Transgenic Pigs ◽  
Human Factor Ix ◽  
Fetal Fibroblasts

Coagulation Factor IX is a vital protein that plays an important role in the blood coagulation cascade. Hereditary deficiency in the Factor IX gene can result in hemophilia type B, the second most common hemophilia. The milk of transgenic livestock can be an efficient vehicle for producing complex, post-translationally modified blood proteins. The advantages include decreased pathogen risk as well as 100-fold or more production efficiency over blood fractionation and cell culture biotechnology. Although the mammary gland can make many of the complex post-translational modifications necessary for biological function of blood proteins, improvements in propeptide cleavage and decreased proteolytic degradation are desirable. To explore the possibility of producing bioactive human Factor IX protein in pig milk, male and female Landrace fetal fibroblast cells were co-transfected by electroporation with 3 different transgene constructs, Factor IX (FIX), Furin, and SERPINA1. The SERPINA1 construct (containing a Neo selectable marker) was delivered into fetal fibroblasts at a 10-fold-lower molar concentration than the other 2 constructs. Following selection in Geneticin, the presence of all 3 genes was verified by PCR and then cells were used as donors for somatic cell nuclear transfer. Thirteen F0 female piglets from 3 potential different integrations were delivered, and 6 piglets were validated by PCR to be positive for all 3 genes. Among the 6 transgenic pigs, 3 are healthy and able to reach puberty. Milk was collected by induced lactation from 2 gilts. A short murine whey acidic protein promoter-Furin gene was used to limit Furin to the lowest levels needed for pro-FIX processing. Furin was expressed to increase propeptide cleavage efficiency, with the result being complete processing of pro-FIX to FIX at ∼0.3 g L–1 pro-FIX. Total FIX levels were ∼1 g L–1. SERPINA1 was also co-expressed at ∼1 g L–1 or more and this serine protease inhibitor did not seem to inhibit furin processing of the pro-FIX. Fifteen F0 male piglets from 3 potential different integration sites were delivered and all of them were positive for all 3 genes. Four F0 males were chosen to breed with wild-type females, and 5 litters of F1 piglets were born. Of 63 F1 piglets, 22 were tri-transgenic and 3 were di-transgenic (only carrying FIX and Furin). Two F1 females were mated with wild-type males, are confirmed to be pregnant, and will be used to determine the expression level and bioactivity of the Factor IX protein in the milk. Funded by the NIH NCRR (RR018877) and R01 HL078944.

scholarly journals Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nis V. Nielsen ◽  
Elfie Roedel ◽  
Dipankar Manna ◽  
Michael Etscheid ◽  
Jens Preben Morth ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Serine Protease ◽  
Active Site ◽  
Factor Vii ◽  
Protease Domain ◽  
Functional Changes ◽  
E Coli ◽  
Catalytic Activation ◽  
Serine Protease Domain ◽  
Tissue Factor Pathway

AbstractFactor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445 ◽  
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Abstract Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

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