Sugar Matters: Improving In Vivo Clearance Rate of Highly Glycosylated Recombinant Plasma Proteins for Therapeutic Use.

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. N-glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA120) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.

SERPING1 mutation update: Mutation spectrum and C1 Inhibitor phenotypes.

C1 inhibitor (C1Inh) deficiency is responsible for hereditary angioedema (C1-INH-HAE) and caused by variants of the SERPING1/C1INH/C1NH gene. C1Inh is the major control of kallikrein-kinin system. C1Inh deficiency leads to its uncontrolled activation, with subsequent generation of the vasoactive peptide bradykinin. This update documents 748 different SERPING1 variants, including published variants and additional 120 unpublished ones. They were identified as heterozygous variants (n = 729), as homozygous variants in 10 probands and as compound heterozygous variants (nine combinations). Six probands with heterozygous variants exhibited gonadal mosaicism. Probands with heterozygous (n = 72) and homozygous (n = 1) variants were identified as de novo cases. Overall, 58 variants were found at positions showing high residue conservation among serpins, and have been referred to as a mousetrap function of C1Inh: reactive center loop, gate, shutter, breach, and hinge. C1Inh phenotype analysis identified dysfunctional serpin variants with failed serpin-protease association and a residual 105-kDa species after incubation with target protease. Regarding this characteristic, in conditions with low antigenic C1Inh, 74 C1-INH-HAE probands presented with an additional so-called intermediate C1-INH-HAE phenotype. The present update addresses a comprehensive SERPING1 variant spectrum that facilitates genotype-phenotype correlations, highlighting residues of strategic importance for serpin function and for identification of C1Inh deficiency as serpinopathy.

Persistent reduction of hippocampal glutamine synthetase expression after status epilepticus in immature rats.

Mesiotemporal sclerosis (MTS), the most frequent form of drug-resistant temporal lobe epilepsy, often develops after an initial precipitating injury affecting the immature brain. To analyse early processes in epileptogenesis we used the juvenile pilocarpine model to study status epilepticus (SE)-induced changes in expression of key components in the glutamate-glutamine cycle, known to be affected in MTS patients. SE was induced by Li(+) /pilocarpine injection in 21-day-old rats. At 2-19 weeks after SE hippocampal protein expression was analysed by immunohistochemistry and neuron damage by FluoroJade staining. Spontaneous seizures occurred in at least 44% of animals 15-18 weeks after SE. As expected in this model, we did not observe loss of principal hippocampal neurons. Neuron damage was most pronounced in the hilus, where we also detected progressive loss of parvalbumin-positive GABAergic interneurons. Hilar neuron loss (or end-folium sclerosis), a common feature in patients with MTS, was accompanied by a progressively decreased glutamine synthetase (GS)-immunoreactivity from 2 (-15%) to 19 weeks (-33.5%) after SE. Immunoreactivity for excitatory amino-acid transporters, vesicular glutamate transporter 1 and glial fibrillary acidic protein was unaffected. Our data show that SE elicited in 21-day-old rats induces a progressive reduction in hilar GS expression without affecting other key components of the glutamate-glutamine cycle. Reduced expression of glial enzyme GS was first detected 2 weeks after SE, and thus clearly before spontaneous recurrent seizures occurred. These results support the hypothesis that reduced GS expression is an early event in the development of hippocampal sclerosis in MTS patients and emphasize the importance of astrocytes in early epileptogenesis.

In vivo MRS and histochemistry of status epilepticus-induced hippocampal pathology in a juvenile model of temporal lobe epilepsy.

Childhood status epilepticus (SE) initiates an epileptogenic process that leads to spontaneous seizures and hippocampal pathology characterized by neuronal loss, gliosis and an imbalance between excitatory and inhibitory neurotransmission. It remains unclear whether these changes are a cause or consequence of chronic epilepsy. In this study, in vivo MRS was used in a post-SE juvenile rat model of temporal lobe epilepsy (TLE) to establish the temporal evolution of hippocampal injury and neurotransmitter imbalance. SE was induced in P21 rats by injection of lithium and pilocarpine. Four and eight weeks after SE, in vivo (1) H and γ-aminobutyric acid (GABA)-edited MRS of the hippocampus was performed in combination with dedicated ex vivo immunohistochemistry for the interpretation and validation of MRS findings. MRS showed a 12% decrease (p<0.0001) in N-acetylaspartate and a 15% increase (p=0.0226) in choline-containing compound concentrations, indicating neuronal death and gliosis, respectively. These results were confirmed by FluoroJade and vimentin staining. Furthermore, severe and progressive decreases in GABA (-41%, p<0.001) and glutamate (Glu) (-17%, p<0.001) were found. The specific severity of GABAergic cell death was confirmed by parvalbumin immunoreactivity (-68%, p<0.001). Unexpectedly, we found changes in glutamine (Gln), the metabolic precursor of both GABA and Glu. Gln increased at 4 weeks (+36%, p<0.001), but returned to control levels at 8 weeks. This decrease was consistent with the simultaneous decrease in glutamine synthase immunoreactivity (-32%, p=0.037). In vivo MRS showed gliosis and (predominantly GABAergic) neuronal loss. In addition, an increase in Gln was detected, accompanied by a decrease in glutamine synthase immunoreactivity. This may reflect glutamine synthase downregulation in order to normalize Gln levels. These changes occurred before spontaneous recurrent seizures were present but, by creating a pre-epileptic state, may play a role in epileptogenesis. MRS can be applied in a clinical setting and may be used as a noninvasive tool to monitor the development of TLE.

Prolonged increase in rat hippocampal chemokine signalling after status epilepticus.

Temporal lobe epilepsy (TLE) is one of the most common focal epilepsy syndromes. In a genome-wide expression study of the human TLE hippocampus we previously showed up-regulation of genes involved in chemokine signalling. Here we investigate in the rat pilocarpine model for TLE, whether changes in chemokine signalling occur during epileptogenesis and are persistent. Therefore we analysed hippocampal protein expression and cellular localisation of CCL2, CCL4, CCR1 and CCR5 after status epilepticus. We found increased CCL4 (but not CCL2) expression in specific populations of hilar astrocytes at 2 and 19 weeks after SE concomitant with a persistent up-regulation of its receptor CCR5. Our results show an early and persistent up-regulation of CCL4/CCR5 signalling during epileptogenesis and suggest that CCL4 signalling, rather than CCL2 signalling, could have a role in the epileptogenic process.

Modulation of the proteolytic activity of the complement protease C1s by polyanions: implications for polyanion-mediated acceleration of interaction between C1s and SERPING1.

The complement system plays crucial roles in the immune system, but incorrect regulation causes inflammation and targeting of self-tissue, leading to diseases such as systemic lupus erythematosus, rheumatoid arthritis and age-related macular degeneration. In vivo, the initiating complexes of the classical complement and lectin pathways are controlled by SERPING1 [(C1 inhibitor) serpin peptidase inhibitor, clade G, member 1], which inactivates the components C1s and MASP-2 (mannan-binding lectin serine peptidase 2). GAGs (glycosaminoglycan) and DXS (dextran sulfate) are able to significantly accelerate SERPING1-mediated inactivation of C1s, the key effector enzyme of the classical C1 complex, although the mechanism is poorly understood. In the present study we have shown that C1s can bind to DXS and heparin and that these polyanions enhanced C1s proteolytic activity at low concentrations and inhibited it at higher concentrations. The recent determination of the crystal structure of SERPING1 has given rise to the hypothesis that both the serpin (serine protease inhibitor)-polyanion and protease-polyanion interactions might be required to accelerate the association rate of SERPING1 and C1s. To determine what proportion of the acceleration was due to protease-polyanion interactions, a chimaeric mutant of alpha1-antitrypsin containing the P4-P1 residues from the SERPING1 RCL (reactive-centre loop) was produced. Like SERPING1, this molecule is able to effectively inhibit C1s, but is unable to bind polyanions. DXS exerted a biphasic effect on the association rate of C1s which correlated strongly with the effect of DXS on C1s proteolytic activity. Thus, whereas polyanions are able to bind C1s and modulate its activity, polyanion interactions with SERPING1 must also play a vital role in the mechanism by which these cofactors accelerate the C1s-SERPING1 reaction.

C1 inhibitor: just a serine protease inhibitor? New and old considerations on therapeutic applications of C1 inhibitor.

C1 inhibitor is a potent anti-inflammatory protein as it is the major inhibitor of proteases of the contact and the complement systems. C1-inhibitor administration is an effective therapy in the treatment of patients with hereditary angioedema (HAE) who are genetically deficient in C1 inhibitor. Owing to its ability to modulate the contact and complement systems and the convincing safety profile, plasma-derived C1 inhibitor is an attractive therapeutic protein to treat inflammatory diseases other than HAE. In the present review we give an overview of the biology of C1 inhibitor and its use in HAE. Furthermore, we discuss C1 inhibitor as an experimental therapy in diseases such as sepsis and myocardial infarction.

Functional C1-inhibitor diagnostics in hereditary angioedema: assay evaluation and recommendations.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition to low C4 levels, the most important laboratory parameter for correct diagnosis of HAE or angioedema due to acquired C1-Inh deficiency is reduced C1-Inh function (fC1-Inh). No direct recommendations about the assays for fC1-Inh or sample handling conditions are available, although this would prove especially useful when a laboratory first starts to offer assays on fC1-Inh for HAE diagnosis. In the present study we evaluated the performance of fC1-Inh assays in the 15 different laboratories that are specialised in HAE diagnostics and assessed inter-laboratory variation with each laboratory using their own assays and standards. A double-blind survey was conducted using plasma/serum samples from healthy donors and HAE patients and the uniformity of HAE diagnosis was evaluated. It can be concluded that the diagnosis of fC1-Inh deficiency was made correctly in most cases in this survey. We can recommend the chromogenic assay for the determination of fC1-Inh, while the complex ELISA needs further investigation.

Soluble complement receptor 1 protects the peripheral nerve from early axon loss after injury.

Complement activation is a crucial early event in Wallerian degeneration. In this study we show that treatment of rats with soluble complement receptor 1 (sCR1), an inhibitor of all complement pathways, blocked both systemic and local complement activation after crush injury of the sciatic nerve. Deposition of membrane attack complex (MAC) in the nerve was inhibited, the nerve was protected from axonal and myelin breakdown at 3 days after injury, and macrophage infiltration and activation was strongly reduced. We show that both classical and alternative complement pathways are activated after acute nerve trauma. Inhibition of the classical pathway by C1 inhibitor (Cetor) diminished, but did not completely block, MAC deposition in the injured nerve, blocked myelin breakdown, inhibited macrophage infiltration, and prevented macrophage activation at 3 days after injury. However, in contrast to sCR1 treatment, early signs of axonal degradation were visible in the nerve, linking MAC deposition to axonal damage. We conclude that sCR1 protects the nerve from early axon loss after injury and propose complement inhibition as a potential therapy for the treatment of diseases in which axon loss is the main cause of disabilities.

Structure and function of C1-inhibitor.

C1-INH belongs to the family of serpins. Structural studies have yielded a clear understanding of the biochemical principle underlying the functional activities of these proteins. Although the crystal structure of C1-INH has yet to be revealed, homology modeling has provided a three-dimensional model of the serpin part of C1-INH. This model has helped us understand the biochemical consequences of mutations of the C1-INH gene as they occur in patients who have HAE. The structure of the N-terminal domain of C1-INH remains unknown; however, this part of the molecule is unlikely to be important in the inhibitory activity of C1-INH toward its target proteases. Mutations in this part have not been described in patients who have HAE, except for a deletion containing two cysteine residues involved in the stabilization of the serpin domain. Recent studies suggest some anti-inflammatory functions for this N-terminal part, possibly explaining the effects of C1-INH in diseases other than HAE.

Effect of reactive site loop elongation on the inhibitory activity of C1-inhibitor.

The serine protease inhibitor C1-Inhibitor (C1-Inh) inhibits several complement- and contact-system proteases, which play an important role in inflammation. C1-Inh has a short reactive site loop (RSL) compared to other serpins. RSL length determines the inhibitory activity of serpins. We investigated the effect of RSL elongation on inhibitory activity of C1-Inh by insertion of one or two alanine residues in the RSL. One of five mutants had an increased association rate with kallikrein, but was nevertheless a poor inhibitor because of a simultaneous high stoichiometry of inhibition (>10). The association rate of the other variants was lower than that of wild-type C1-Inh. These data suggest that the relatively weak inhibitory activity of C1-Inh is not the result of its short RSL. The short RSL of C1-Inh has, surprisingly, the optimal length for inhibition.

Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor.

Therapeutic application of the serpin C1-inhibitor (C1-Inh) in inflammatory diseases like sepsis, acute myocardial infarction and vascular leakage syndrome seems promising, but large doses may be required. Therefore, a high-yield recombinant expression system for C1-Inh is very interesting. Earlier attempts to produce high levels of C1-Inh resulted in predominantly inactive C1-Inh. We describe the high yield expression of rhC1-Inh in Pichia pastoris, with 180 mg/l active C1-Inh at maximum. On average, 30 mg/l of 80-100% active C1-Inh was obtained. Progress curves were used to study the interaction with C1s, kallikrein, coagulation factor XIIa and XIa, and demonstrated that rhC1-Inh had the same inhibitory capacity as plasma C1-Inh. Structural integrity, as monitored via heat stability, was comparable despite differences in extent and nature of glycosylation. We conclude that the P. pastoris system is capable of high-level production of functionally and structurally intact human C1 inhibitor.

The functional integrity of the serpin domain of C1-inhibitor depends on the unique N-terminal domain, as revealed by a pathological mutant.

C1-inhibitor (C1-Inh) is a serine protease inhibitor (serpin) with a unique, non-conserved N-terminal domain of unknown function. Genetic deficiency of C1-Inh causes hereditary angioedema. A novel type of mutation (Delta 3) in exon 3 of the C1-Inh gene, resulting in deletion of Asp62-Thr116 in this unique domain, was encountered in a hereditary angioedema pedigree. Because the domain is supposedly not essential for inhibitory activity, the unexpected loss-of-function of this deletion mutant was further investigated. The Delta 3 mutant and three additional mutants starting at Pro76, Gly98, and Ser115, lacking increasing parts of the N-terminal domain, were produced recombinantly. C1-Inh76 and C1-Inh98 retained normal conformation and interaction kinetics with target proteases. In contrast, C1-Inh115 and Delta 3, which both lack the connection between the serpin and the non-serpin domain via two disulfide bridges, were completely non-functional because of a complex-like and multimeric conformation, as demonstrated by several criteria. The Delta 3 mutant also circulated in multimeric form in plasma from affected family members. The C1-Inh mutant reported here is unique in that deletion of an entire amino acid stretch from a domain not shared by other serpins leads to a loss-of-function. The deletion in the unique N-terminal domain results in a "multimerization phenotype" of C1-Inh, because of diminished stability of the central beta-sheet. This phenotype, as well as the location of the disulfide bridges between the serpin and the non-serpin domain of C1-Inh, suggests that the function of the N-terminal region may be similar to one of the effects of heparin in antithrombin III, maintenance of the metastable serpin conformation.

Cytomegalovirus infection modulates cellular immunity in an experimental model for autoimmune diabetes.

BACKGROUND: Viral infections are thought to play a role in the development of autoimmune diseases like type 1 diabetes. In this study we investigated the effect of Rat Cytomegalovirus (RCMV) infection on cellular immunity in a well-defined animal model for diabetes, the Biobreeding (BB) rat. METHODS: Diabetes prone (DP)- and Diabetes resistant (DR)-BB rats were infected with 2 x 10(6) plaque forming units (pfu) RCMV. Diabetes development was monitored by frequent blood-glucose analysis. Effects of RCMV on CD4+, CD8+ and Vbeta-TCR+ T-cell subsets were measured in vivo, and in vitro after restimulation with RCMV-infected fibroblasts. Proliferative capacity was determined by 3H-Thymidine incorporation. RESULTS: RCMV-infection resulted in a significant acceleration of diabetes onset in DP-BB rats (p = 0.003). Percentages CD4+ and CD8+ T-cells were not affected in vivo. In vitro, RCMV-restimulation resulted in a decreased CD4+/CD8+ blastoid T-cell ratio compared to ConA (p = 0.00028). Furthermore, RCMV-restimulation resulted in a strong RCMV-specific proliferation, which comprises about 50% of the response triggered by ConA. Vbeta-TCR percentages did not change upon RCMV-infection or RCMV-restimulation. INTERPRETATION: RCMV-restimulation of splenic T-cells in vitro resulted in a strong RCMV-specific proliferation, probably also including autoreactive T-cells. In vivo, this polyclonal response might be involved in the observed accelerated diabetes development in DP-BB rats upon RCMV-infection.

Structural and functional aspects of C1-inhibitor.

C1-Inh is a serpin that inhibits serine proteases from the complement and the coagulation pathway. C1-Inh consists of a serpin domain and a unique N-terminal domain and is heavily glycosylated. Non-functional mutants of C1-Inh can give insight into the inhibitory mechanism of C1-Inh. This review describes a novel 3D model of C1-Inh, based on a newly developed homology modelling method. This model gives insight into a possible potentiation mechanism of C1-Inh and based on this model the essential residues for efficient inhibition by C1-Inh are discussed.

Role of complement in graft rejection after organ transplantation.

Activation of the complement system may significantly contribute to the inflammatory reaction after solid organ transplantation. In allotransplantation, the complement system may be activated by ischemia/reperfusion and, possibly, by antibodies directed against the graft. In xenotransplantation from nonprimates to primates, the major activators for complement are preexisting antibodies. Studies in animal models have shown that the use of complement inhibitors may significantly prolong graft survival. This review describes the role of the complement system in organ injury after organ transplantation and the use of complement inhibitors to prevent damage to the graft after allo- or xenotransplantation.