Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.     

Clinical relevance of protein C

Protein C is, after activation by thrombin, a potent inhibitor of blood coagulation. An isolated deficiency of protein C increases the risk of thrombosis. The two forms of protein C deficiency, the heterozygous and the homozygous deficiency state, have different clinical features. Patients with heterozygous protein C deficiency are at a high risk to develop venous thrombosis and pulmonary embolism. In newborns with homozygous protein C deficiency with very low protein C levels (1%) a purpura fulminans like syndrome was observed. Heparin and coumarin derivatives are effective drugs in heterozygous protein C deficiency, homozygous patients may be treated either by replacement of protein C or coumarin derivatives. Decreased protein C levels were observed in various other diseases: Chronic and acute liver disease, disseminated intravascular coagulation, malignancy, postoperatively and during treatment with asparaginase. The role of protein C in these diseases to trigger thrombosis is not yet established.     

Complement regulatory protein C1 inhibitor binds to selectins and interferes with endothelial-leukocyte adhesion

C1 inhibitor (C1INH), a member of the serine proteinase inhibitor (serpin) family, is an inhibitor of proteases in the complement system, the contact system of kinin generation, and the intrinsic coagulation pathway. It is the most heavily glycosylated plasma protein, containing 13 definitively identified glycosylation sites as well as an additional 7 potential glycosylation sites. C1INH consists of two distinct domains: a serpin domain and an amino-terminal domain. The serpin domain retains all the protease-inhibitory function, while the amino-terminal domain bears most of the glycosylation sites. The present studies test the hypothesis that plasma C1INH bears sialyl Lewis(x)-related moieties and therefore binds to selectin adhesion molecules. We demonstrated that plasma C1INH does express sialyl Lewis(x)-related moieties on its N-glycan as detected using mAb HECA-452 and CSLEX1. The data also show that plasma C1INH can bind to P- and E-selectins by FACS and immunoprecipitation experiments. In a tissue culture model of endothelial-leukocyte adhesion, C1INH showed inhibition in a dose-dependent manner. Significant inhibition (>50%) was achieved at a concentration of 250 micro g/ml or higher. This discovery may suggest that C1INH plays a role in the endothelial-leukocyte interaction during inflammation. It may also provide another example of the multifaceted anti-inflammatory effects of C1INH in various animal models and human diseases.     

Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits

The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found.     

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.     

An anti-endotoxin peptide that generates from the amino-terminal domain of complement regulatory protein C1 inhibitor

C1 inhibitor (C1INH), a complement regulatory protein, prevents endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial lipopolysaccharide (LPS). Importantly, the cleaved, inactive C1INH still is an anti-endotoxin effector indicating the anti-endotoxin peptide that generates from the amino-terminal domain of C1INH. In this study, we first identified that a cleaved fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of C1INH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH cleaved fragment. Among these synthetic peptides, a 13-mer derivative peptide at position from 18 to 30, named N2((18-30)), exhibited the most powerful anti-endotoxin activity in vitro, enlightening that it was most strong at binding to LPS, inhibiting the interaction of LPS with LPS-binding protein (LBP), blocking LPS binding to CD14(+) cells, and suppressing production of tumor necrosis factor (TNF)-alpha by murine macrophages, RAW 264.7. In the murine endotoxin shock model, the peptide N2((18-30)) protected mice from LPS-induced lethal septic shock by inhibiting macrophage activation. These data indicate that the peptide N2((18-30)) derived from the amino-terminal region of C1INH is anti-endotoxin.     

Two different missense mutations at Arg 178 of the protein C (PROC) gene causing recurrent venous thrombosis

Summary Non-identical missense mutations were identified at Arg 178 in the protein C genes of two patients with heterozygous type 1 protein C deficiency and recurrent venous thrombosis.     

Heterozygous protein C deficiency type I

Protein C is a vitamin K-dependent plasma protein which has anticoagulatory and profibrinolytic properties as a result of inactivating coagulation factors Va and VIIIa and enhancing fibrinolysis. Heterozygous protein C deficiency is well known to be a risk factor for thromboembolic diseases. We here present a family with 16 members deficient in protein C, out of which only two persons were suffering from thromboembolic disorders. In patients suffering from heterozygous protein C deficiency thromboembolic complications in childhood are rare and are not obligatory in adults. These patients should therefore not be treated with oral anticoagulants unless thromboembolic complications have already occurred or are imminent. Coumarin anticoagulation implicates a serious risk of coumarin skin necrosis in protein C deficient patients during the initial therapeutic phase. This risk may be avoided by initiating coumarin therapy with low doses of the drug and in cases of thromboembolic complications by overlapping with heparin anticoagulation.     

C1 inhibitor prevents endotoxin shock via a direct interaction with lipopolysaccharide

C1 inhibitor (C1INH) is beneficial in animal models of endotoxemia and sepsis. However, the mechanism(s) of C1INH protection remain(s) ill-defined. In this study, we demonstrated that both active C1INH and reactive center-cleaved, inactive C1INH protected mice from lethal Gram-negative endotoxemia. Both forms of C1INH blocked the LPS-binding protein-dependent binding of Salmonella typhimurium LPS to the murine macrophage cell line, RAW 264.7, and suppressed LPS-induced TNF-alpha mRNA expression. Inhibition of LPS binding to RAW 264.7 cells was reversed with anti-C1INH Ab and was more efficient when C1INH was incubated first with LPS rather than with the cells. C1INH also suppressed LPS-induced up-regulation of TNF-alpha mRNA in whole human blood. The interaction of C1INH with LPS was directly demonstrated both by ELISA and by nondenaturing PAGE, but deletion of the amino-terminal 97-aa residues abrogated this binding. Therefore, C1INH, in addition to its function as a serine protease inhibitor, has a novel anti-inflammatory function mediated via its heavily glycosylated amino-terminal non-serpin domain.     

N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits

Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.     

Mutational spectrum of the C1INH (SERPING1) gene in patients with hereditary angioedema

Hereditary angioedema (HAE) is an autosomal dominant disease that manifests as intermittent acute swellings of the skin and mucosal surfaces, which, in the gastrointestinal tract and larynx, may even be fatal. HAE results from functional deficiency of the C1 inhibitor (C1INH) protein, which plays a key role in the classical pathway of complement activation. C1INH is the sole inhibitor of the activated proteases C1r and C1s, and is the major regulator of activated coagulation Factor XII and plasma kallikrein, which limits the generation of the vasoactive peptide bradykinin. In this paper, we report on the genetic analysis of 173 families (including 326 members) with a clinical diagnosis of HAE. Direct sequencing, Southern blotting and quantitative PCR by the MLPA method were used to screen for mutations in C1INH (SERPING1). In 142 families (82.1%), a causative C1INH gene mutation could be identified. A total of 80 novel point mutations of C1INH not published previously were detected in 96 pedigrees (including 172 members). Our results corroborate C1INH (SERPING1) deficiency as a disease of extreme allelic heterogeneity with almost each individual family carrying their own mutation. Routine molecular genetic analysis is an effective way of confirming the clinical diagnosis and identifying mutation carriers early on before any clinical manifestation becomes apparent. It is, therefore, a valuable tool in prevention and adequate treatment of acute and life-threatening oedema.     

Mutational spectrum of D-bifunctional protein deficiency and structure-based genotype-phenotype analysis

D-bifunctional protein (DBP) deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. The clinical presentation of DBP deficiency is usually very severe, but a few patients with a relatively mild presentation have been identified. In this article, we report the mutational spectrum of DBP deficiency on the basis of molecular analysis in 110 patients. We identified 61 different mutations by DBP cDNA analysis, 48 of which have not been reported previously. The predicted effects of the different disease-causing amino acid changes on protein structure were determined using the crystal structures of the (3R)-hydroxyacyl-coenzyme A (CoA) dehydrogenase unit of rat DBP and the 2-enoyl-CoA hydratase 2 unit and liganded sterol carrier protein 2-like unit of human DBP. The effects ranged from the replacement of catalytic amino acid residues or residues in direct contact with the substrate or cofactor to disturbances of protein folding or dimerization of the subunits. To study whether there is a genotype-phenotype correlation for DBP deficiency, these structure-based analyses were combined with extensive biochemical analyses of patient material (cultured skin fibroblasts and plasma) and available clinical information on the patients. We found that the effect of the mutations identified in patients with a relatively mild clinical and biochemical presentation was less detrimental to the protein structure than the effect of mutations identified in those with a very severe presentation. These results suggest that the amount of residual DBP activity correlates with the severity of the phenotype. From our data, we conclude that, on the basis of the predicted effect of the mutations on protein structure, a genotype-phenotype correlation exists for DBP deficiency.     

Ongoing Contact Activation in Patients with Hereditary Angioedema

Hereditary angioedema (HAE) is predominantly caused by a deficiency in C1 esterase inhibitor (C1INH) (HAE-C1INH). C1INH inhibits activated factor XII (FXIIa), activated factor XI (FXIa), and kallikrein. In HAE-C1INH patients the thrombotic risk is not increased even though activation of the contact system is poorly regulated. Therefore, we hypothesized that contact activation preferentially leads to kallikrein formation and less to activation of the coagulation cascade in HAE-C1INH patients. We measured the levels of C1INH in complex with activated contact factors in plasma samples of HAE-C1INH patients (N=30, 17 during remission and 13 during acute attack) and healthy controls (N=10). We did not detect differences in enzyme-inhibitor complexes between samples of controls, patients during remission and patients during an acute attack. Reconstitution with C1INH did not change this result. Next, we determined the potential to form enzyme-inhibitory complexes after complete in vitro activation of the plasma samples with a FXII trigger. In all samples, enzyme-C1INH levels increased after activation even in patients during an acute attack. However, the levels of FXIIa-C1INH, FXIa-C1INH and kallikrein-C1INH were at least 52% lower in samples taken during remission and 70% lower in samples taken during attack compared to samples from controls (p<0.05). Addition of C1INH after activation led to an increase in levels of FXIIa-C1INH and FXIa-C1INH (p<0.05), which were still lower than in controls (p<0.05), while the levels of kallikrein-C1INH did not change. These results are consistent with constitutive activation and attenuated depletion of the contact system and show that the ongoing activation of the contact system, which is present in HAE-C1INH patients both during remission and during acute attacks, is not associated with preferential generation of kallikrein over FXIa.     

Biosynthesis of a structurally abnormal C2 complement protein by macrophages from C2-deficient guinea pigs

In order to characterize a genetic deficiency of C2 in guinea pigs, production of C2 by peritoneal macrophage cultures derived from four normal, four heterozygous deficient, and four homozygous deficient animals was measured functionally and immunochemically after metabolic labeling with 35S-methionine. Macrophage monolayers from homozygous deficient animals failed to secrete hemolytically detectable C2 up to 74 hr in culture. A single cell hemolytic plaque assay also failed to demonstrate any functional C2 production by cells from homozygous deficient animals. No C2 protein was detected in media from three of the four homozygous deficient animals, but in one, apparent C2 fragments were present. In contrast, intracellular C2 protein was identified in all four homozygous deficient cell cultures. Its mobility on SDS-PAGE was slightly faster than normal. Much less abnormal intracellular C2 protein was recovered from homozygous deficient macrophage monolayers than intracellular C2 protein from normal macrophage monolayers. Monolayers from heterozygous animals produced functional and immunochemical C2 at approximately 30% of the normal rate. Normal rates of biosynthesis and secretion of two other MHC-linked class III antigens, C4 and factor B, were detected in macrophage cultures from homozygous and heterozygous deficient animals. These data suggest that a specific defect, i.e. a structural abnormality in C2 protein, underlies C2 deficiency in guinea pigs.     

Interferon-gamma is a major regulator of C1-inhibitor synthesis by human blood monocytes

C1 inhibitor (C1INH) is the major control factor for the activation of the classical pathway of complement and for contact system activation. Hepatocytes and blood monocytes are known to synthesize this protease inhibitor. We studied the regulation of monocyte C1INH production by mediators that are generated during inflammatory responses. Purified blood monocytes spontaneously synthesized and secreted C1INH only after prolonged culture. In the presence of interferon (IFN)-gamma, C1INH was detectable within 24 hr and continued to be released at high levels throughout an 8-day culture period. Monocyte C1INH was newly synthesized and was functionally active as determined by forming stable complex with C1s. Other monocyte stimuli were either less potent (IFN-alpha, IFN-beta) or not capable of increasing C1INH release (lipopolysaccharide, interleukin 1, and tumor necrosis factor). The second component of complement, C2, was induced by IFN-gamma to a similar extent as C1INH. These findings demonstrate that IFN-gamma is a major regulator of monocyte C1INH production and may warrant consideration of IFN-gamma in the treatment of C1INH deficiency states.     

Golgi alpha-mannosidase II deficiency in vertebrate systems: implications for asparagine-linked oligosaccharide processing in mammals

The maturation of N-glycans to complex type structures on cellular and secreted proteins is essential for the roles that these structures play in cell adhesion and recognition events in metazoan organisms. Critical steps in the biosynthetic pathway leading from high mannose to complex structures include the trimming of mannose residues by processing mannosidases in the endoplasmic reticulum (ER) and Golgi complex. These exo-mannosidases comprise two separate families of enzymes that are distinguished by enzymatic characteristics and sequence similarity. Members of the Class 2 mannosidase family (glycosylhydrolase family 38) include enzymes involved in trimming reactions in N-glycan maturation in the Golgi complex (Golgi mannosidase II) as well as catabolic enzymes in lysosomes and cytosol. Studies on the biological roles of complex type N-glycans have employed a variety of strategies including the treatment of cells with glycosidase inhibitors, characterization of human patients with enzymatic defects in processing enzymes, and generation of mouse models for the enzyme deficiency by selective gene disruption approaches. Corresponding studies on Golgi mannosidase II have employed swainsonine, an alkaloid natural plant product that causes "locoism", a phenocopy of the lysosomal storage disease, alpha-mannosidosis, as a result of the additional targeting of the broad-specificity lysosomal mannosidase by this compound. The human deficiency in Golgi mannosidase II is characterized by congenital dyserythropoietic anemia with splenomegaly and various additional abnormalities and complications. Mouse models for Golgi mannosidase II deficiency recapitulate many of the pathological features of the human disease and confirm that the unexpectedly mild effects of the enzyme deficiency result from a tissue-specific and glycoprotein substrate-specific alternate pathway for synthesis of complex N-glycans. In addition, the mutant mice develop symptoms of a systemic autoimmune disorder as a consequence of the altered glycosylation. This review will discuss the biochemical features of Golgi mannosidase II and the consequences of its deficiency in mammalian systems as a model for the effects of alterations in vertebrate N-glycan maturation during development.     

A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.     

Coumarin induced acral skin necrosis associated with hereditary protein C deficiency

Hemorrhagic skin necrosis of the toes was observed in a patient with heterozygous protein C deficiency (protein C:Ag 32% and protein C activity 30%) on the 4th day of coumarin treatment overlapping with effective intravenous anticoagulation with heparin. Family studies revealed protein C deficiency in two sisters of the proposita without a history of thromboembolic disease. Immunologic studies in the proposita at the time of coumarin necrosis revealed slight depression of complement factor C4 and the presence of immune complexes. The present case and review of the literature show that the pathogenetic mechanism leading to coumarin necrosis in patients with protein C deficiency seems not yet to be fully understood.     

A direct role for C1 inhibitor in regulation of leukocyte adhesion

Plasma C1 inhibitor (C1INH) is a natural inhibitor of complement and contact system proteases. Heterozygosity for C1INH deficiency results in hereditary angioedema, which is mediated by bradykinin. Treatment with plasma C1INH is effective not only in patients with hereditary angioedema, but also in a variety of other disease models, in which such therapy is accompanied by diminished neutrophil infiltration. The underlying mechanism has been explained primarily as a result of the inhibition of the complement and contact systems. We have shown that C1INH expresses the sialyl-Lewis(x) tetrasaccharide on its N-linked glycan, via which it binds to E- and P-selectins and interferes with leukocyte-endothelial adhesion in vitro. Here we show that both native C1INH and reactive center cleaved C1INH significantly inhibit selectin-mediated leukocyte adhesion in several in vitro and in vivo models, whereas N-deglycosylated C1INH loses such activities. The data support the hypothesis that C1INH plays a direct role in leukocyte-endothelial cell adhesion, that the activity is mediated by carbohydrate, and that it is independent of protease inhibitory activity. Direct involvement of C1INH in modulation of selectin-mediated cell adhesion may be an important mechanism in the physiologic suppression of inflammation, and may partially explain its utility in therapy of inflammatory diseases.