A new simplified procedure for C1 inhibitor purification: A novel use for jacalin-agarose

doi:10.1016/0022-1759(89)90286-X

Journal of Immunological Methods

Volume 120, Issue 1, 2 June 1989, Pages 37-43

https://doi.org/10.1016/0022-1759(89)90286-X Get rights and content

Abstract

C1 inhibitor (C1-INH), the major regulatory protein of the classical pathway of complement activation, is also involved in the regulation of several other plasma proteolytic systems including the coagulation, fibrinolytic and contact systems. All the previously published methods for the purification of C1-INH are time-consuming and some do not yield highly pure protein. Recently, it was reported that Jack fruit (Artocarpus integrifolia) lectin, also called jacalin, binds C1-INH. Since jacalin binds only a small number of human serum proteins it appeared that jacalin-agarose affinity chromatography would constitute a very selective early step for the purification of C1-INH. Consequently we have designed a new, simplified three-step procedure for the purification of C1-INH which includes PEG fractionation, jacalin-agarose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose which takes advantage of the marked hydrophilicity of the inhibitor. This procedure has three major advantages over those which have been the most frequently used. Firstly, it includes only two fast chromatographic steps. Secondly, because the C1-INH pool is cleanly and predictably separated from the unwanted proteins by differential elution conditions in both chromatographic steps, no antigenic or functional assays are required to define the desired peaks. Thirdly, only the final product is dialyzed while all other methods required several buffer changes. For these reasons this procedure is much faster and simpler than the previously published methods. About 10–12 mg of highly purified and fully active C1-INH can be obtained within 1 day from 120 ml of plasma giving an average yield of 40–45%. This method may thus be highly adaptable to bulk purification for clinical use or for preparation of genetically or pathologically altered C1-INH from clinical specimens.

References (20)

N.R. Cooper
The classical complement pathway: Activation and regulation of the first complement component
Adv. Immunol.
(1985)
R.L. Gregory et al.
Separation of human IgA1 and IgA2 using jacalin-agarose chromatography
J. Immunol. Methods
(1987)
K. Hagiwara et al.
Jacalin: Isolation, characterization and influence of various factors on its interaction with human IgA as assessed by precipitation and latex agglutination
Mol. Immunol.
(1988)
J.V. Maizel
Polyacrylamide gel electrophoresis of viral proteins
A. Malbran et al.
Acquired angioedema: Observations on the mechanism of action of autoantibodies directed against C1-esterase inhibitor
J. Allerg. Clin. Immunol.
(1988)
G. Mancini et al.
Immunochemical quantitation of antigens by single radial immunodiffusion
Immunochemistry
(1965)
L.G. Prograis et al.
Purification of C1-inhibitor. A new approach for the isolation of this biologically important plasma protease inhibitor
J. Immunol. Methods
(1987)
A. Reboul et al.
A simplified method for the purification of C1-inactivator from human plasma. Interaction with complement subcomponents C1r and C1s
FEBS Lett.
(1977)
D.L. Skea et al.
Studies on the specificity of the IgA-binding lectin, jacalin
Mol. Immunol.
(1988)
J. Alsenz et al.
Autoantibody-mediated acquired deficiency of C1-inhibitor
New Engl. J. Med.
(1987)

There are more references available in the full text version of this article.

Cited by (54)

Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits
2012, Journal of Biotechnology
Citation Excerpt :
The supernatant was loaded on a SP Sepharose column (1.6/15 cm) and unbound proteins were removed by washing with 20 mM sodium phosphate pH 7.0. Bound proteins were eluted with 20 mM sodium phosphate, 0.5 M NaCl of pH 7.0 and subjected to Jacalin agarose column (1.6/5 cm) chromatography as described (Pilatte et al., 1989). The eluate of the Jacalin agarose column was diluted with 20 mM sodium phosphate pH 5.5 and loaded on a Q Sepharose column (0.46/15 cm) equilibrated in 20 mM sodium phosphate, 50 mM NaCl of pH 5.5.
C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5′ bovine αS₁ casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M_r) of 67,200. Differences in M_r on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest^®) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE.
Multiple domains of MASP-2, an initiating complement protease, are required for interaction with its substrate C4
2012, Molecular Immunology
Citation Excerpt :
Commercial PCR clean-up kits were purchased from Millipore (Billerica, MA, USA). C1-inhibitor (C1-Inh) was purified from human plasma as described previously (Pilatte et al., 1989). Human C4 and C4b were purchased from CompTech (Tyler, Texas, USA).
The complement system is fundamental to both innate and adaptive immunity and can be initiated via the classical, lectin or alternative pathways. Cleavage of C4 by MASP-2, the initiating protease of the lectin pathway, is a crucial event in the activation of this pathway, preceding the eventual formation of the C3 convertase (C4bC2a) complex on the pathogen surface. Interactions required for the cleavage of C4 by MASP-2 are likely to be facilitated by the initial binding of C4 to an exosite on the protease. We have shown that both proteolytically active and catalytically inactive CCP1–CCP2–serine protease (CCP1–CCP2–SP) forms bind C4 with similar affinity. Interestingly, proteins containing the CCP1–CCP2 domains or the SP domain alone bound C4 with much lower affinity than the CCP1–CCP2–SP protein, suggesting that the CCP domains cooperate positively with the active site to mediate efficient binding and cleavage of C4. In addition, mutation of residue K342 to alanine in the CCP1 domain abolished binding to both C4 and C4b in its CCP1–CCP2 form, suggesting a key electrostatic role for this amino acid. The presented data indicates that all of the domains are required in order to mediate high affinity interaction with C4.
Serpins and the complement system
2011, Methods in Enzymology
Citation Excerpt :
Jacalin is a d-galactose-specific lectin from the jackfruit, Artocarous integrifolia that interacts with only a few serum proteins and has been used to isolate functionally active C1-inhibitor without any degradation (Hiemstra et al., 1987); the presence of a relatively high concentration of salt (0.5 M NaCl) in all the buffers also minimizes nonspecific binding. C1-inhibitor is markedly hydrophilic protein (Pilatte et al., 1989), thus under the conditions employed, C1-inhibitor is the only protein not retained by the phenyl-sepharose column. This procedure has major advantages over those used previously, first, because it includes only two chromatographic steps.
C1-inhibitor (serpin G1) is a 105 kDa inhibitor which functions as a major antiinflammatory protein in the body. It has its effects via inhibition of the proteases of the complement system and contact system of coagulation, as well as several direct effects mediated by its unique highly glycosylated N-terminal domain. The serpin controls a number of different proteases very efficiently and for some of these the function is augmented by the cofactor, heparin. Here, we describe the preparation of human plasma and recombinant C1-inhibitor and the basic methods required for their characterization, using the complement enzyme C1s as an example of a target enzyme.
Artocarpus: A review of its traditional uses, phytochemistry and pharmacology
2010, Journal of Ethnopharmacology
The genus Artocarpus (Moraceae) comprises about 50 species of evergreen and deciduous trees. Economically, the genus is of appreciable importance as a source of edible fruit, yield fairly good timber and is widely used in folk medicines. The aim of the present review is to present comprehensive information of the chemical constituents, biological and pharmacological research on Artocarpus which will be presented and critically evaluated. The close connection between traditional and modern sources for ethnopharmacological uses of Artocarpus species, especially for treatment against inflammation, malarial fever, diarrhoea, diabetes and tapeworm infection. Artocarpus species are rich in phenolic compounds including flavonoids, stilbenoids, arylbenzofurons and Jacalin, a lectin. The extracts and metabolites of Artocarpus particularly those from leaves, bark, stem and fruit possess several useful bioactive compounds and recently additional data are available on exploitation of these compounds in the various biological activities including antibacterial, antitubercular, antiviral, antifungal, antiplatelet, antiarthritic, tyrosinase inhibitory and cytotoxicity. Several pharmacological studies of the natural products from Artocarpus have conclusively established their mode of action in treatment of various diseases and other health benefits. Jacalin, a lectin present in seeds of this plant has a wide range of activities. Strong interdisciplinary programmes that incorporate conventional and new technologies will be critical for the future development of Artocarpus as a promising source of medicinal products. In the present review, attempts on the important findings have been made on identification; synthesis and bioactivity of metabolites present in Artocarpus which have been highlighted along with the current trends in research on Artocarpus.
Elucidation of the substrate specificity of the MASP-2 protease of the lectin complement pathway and identification of the enzyme as a major physiological target of the serpin, C1-inhibitor
2008, Molecular Immunology
Complement is a central component of host defence, but unregulated activation can contribute to disease. The system can be initiated by three pathways: classical, alternative and lectin. The classical and lectin pathways are initiated by the C1 and mannose-binding lectin (MBL) or ficolin complexes, respectively, with C1s the executioner protease of the C1 complex and MASP-2 its counterpart in the lectin complexes. These proteases in turn cleave the C4 and C2 components of the system. Here we have elucidated the cleavage specificity of MASP-2 using a randomised substrate phage display library. Apart from the crucial P₁ position, the MASP-2 S₂ and S₃ subsites (in that order) play the greatest role in determining specificity, with Gly residues preferred at P₂ and Leu or hydrophobic residues at P₃. Cleavage of peptide substrates representing the known physiological cleavage sequences in C2, C4 or the serpin C1-inhibitor (a likely regulator of MASP-2) revealed that MASP-2 is up to 1000 times more catalytically active than C1s. C1-inhibitor inhibited MASP-2 50-fold faster than C1s and much faster than any other protease tested to date, implying that MASP-2 is a major physiological target of C1-inhibitor.
Kinetic studies on the interactions of heparin and complement proteins using surface plasmon resonance
2005, Biochimica et Biophysica Acta - General Subjects
Heparin is a naturally occurring polysaccharide known to interact with complement proteins and regulate multiple steps in the complement cascade. Quantitative information, in the form of affinity constants for heparin-complement interactions, is not generally available and there are no reports of a comprehensive analysis using the same interaction method. Such information should improve our understanding of how exogenously administered pharmaceutical heparin and the related endogenous polysaccharide, heparan sulfate, regulate complement activation. The current study provides the first comprehensively analysis of the binding of various complement proteins to heparin using surface plasmon resonance (SPR). Complement proteins C1, C2, C3, C4, C5, C6, C7, C8, C9, C1INH, factor I, factor H, factor B and factor P all bind heparin but exhibit different binding kinetics and dissociation constants (K_d) ranging from 2 to 320 nM. By taking into account these K_d values and the serum concentrations of these complement proteins, the percentage of each binding to exogenously administered heparin was calculated and found to range from 2% to 41%. This study provides essential information required for the rational design of new therapeutic agents capable of regulating the complement activation.

View all citing articles on Scopus

View full text

A new simplified procedure for C1 inhibitor purification: A novel use for jacalin-agarose

Abstract

Adv. Immunol.

J. Immunol. Methods

Mol. Immunol.

J. Allerg. Clin. Immunol.

Immunochemistry

J. Immunol. Methods

FEBS Lett.

Mol. Immunol.

Autoantibody-mediated acquired deficiency of C1-inhibitor

New Engl. J. Med.