PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 12893820

Mutations in alpha-chain of C4BP that selectively affect its factor I cofactor function.

Anna M Blom·Bruno O Villoutreix·Björn Dahlbäck
View original source ↗Export metadata ↓

Abstract

C4b-binding protein (C4BP) inhibits all pathways of complement activation, acting as a cofactor to the serine protease factor I (FI) in the degradation of activated complement factors C4b and C3b. C4BP is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain, the alpha- and beta-chains being composed of eight and three complement control protein (CCP) domains, respectively. In previous studies we have localized cofactor activity and binding of C4b to alpha-chain CCP1-3 of C4BP, whereas the binding of C3b required additionally CCP4. Likewise, introduced point mutations that decreased binding of C4b/C3b caused a decrease in cofactor activity. In the present study, we describe two mutants of C4BP, K126Q/K128Q and F144S/F149S, clustered on alpha-chain CCP3, which selectively lost their ability to act as cofactors in the cleavage of both C4b and C3b. Both mutants show the same binding affinity for C4b/C3b as measured by surface plasmon resonance and have the same inhibitory effect on formation and decay of the classical pathway C3-convertase as the wild type C4BP. It appears that C4b and C3b do not undergo the same conformational changes upon binding to the C4BP mutants as during the interaction with the wild type C4BP, which then results in the observed loss of the cofactor activity.

Explore related subjects

Anilino NaphthalenesulfonatesBinding, CompetitiveCircular DichroismComplement ActivationComplement C3bComplement C4bComplement Inactivator ProteinsDNA, ComplementaryDisulfidesDose-Response Relationship, DrugElectrophoresis, Polyacrylamide GelFluorescent DyesGlycoproteinsHumansKineticsMutagenesis, Site-DirectedMutationPoint MutationProtein BindingProtein ConformationProtein Structure, SecondaryRecombinant ProteinsSurface Plasmon ResonanceJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Anna M Blom, Bruno O Villoutreix, Björn Dahlbäck. 2003-07-31. Mutations in alpha-chain of C4BP that selectively affect its factor I cofactor function.. https://doi.org/10.1074/jbc.m306620200

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Biological polyamines inhibit nucleic-acid-induced polymerisation of prion protein.

Nucleic-acid-induced polymerisation of prion protein, when monitored by anilino naphthalene sulfonic acid dye, shows, successively, an immediate fluorescence increase of the dye upon mixing of the reactants, followed by a lag period in which the dye fluorescence remains unchanged, and then a phase in which dye fluorescence increases with time. The biological polyamines spermine and spermidine reduce the extent of the initial fluorescence increase, increase the lag period, and reduce both the rate and the extent of increase in fluorescence intensity of the dye in the final phase of the reaction. Spermidine is less effective than spermine in all of these processes. A nearly fivefold lower concentration of spermine can inhibit polymerisation of prion protein by tRNAs compared to the same process induced by double-stranded nucleic acid. The change in the secondary structure of the globular domain of the protein induced by nucleic acid is reversed by the addition of spermine, and it prevents structural destabilization of this domain induced by nucleic acids. It is suggested that physiological event(s) that would reduce the concentrations of intracellular biological amines may make nucleic acid available to induce oligomerization and polymerisation of cellular prion protein related to prion disease.

Anilino Naphthalenesulfonates↗
PubMed2006

pH-dependent aggregation of cutinase is efficiently suppressed by 1,8-ANS.

We have studied the thermal stability of the triglyceride-hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 microM decreases the apparent melting temperature by 19 degrees C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes, the hydrophobic fluorophore 1,8-ANS completely and saturably suppresses this effect, restoring 70% of enzymatic activity upon cooling. ANS binds strongly to native cutinase as a noncompetitive inhibitor with up to 5 ANS per cutinase molecule. Only the first ANS molecule stabilizes cutinase; however, the last 4 ANS molecules decrease Tm by up to 7 degrees C. Similar pI-dependent aggregation and suppression by ANS is observed for T. lanuginosus lipase, but not for lysozyme or porcine alpha-amylase, suggesting that this behavior is most prevalent for proteins with affinity for hydrophobic substrates and consequent exposure of hydrophobic patches. Aggregation may be promoted by a fluctuating ensemble of native-like states associating via intermolecular beta-sheet rich structures unless blocked by ANS. Our data highlight the chaperone activity of small molecules with affinity for hydrophobic surfaces and their potential application as stabilizers at appropriate stoichiometries.

Anilino Naphthalenesulfonates↗
PubMed2006

Identification and characterization of functional intermediates of stem bromelain during urea and guanidine hydrochloride unfolding.

By comparing changes in enzyme activity with changes in spectral features for stem bromelain (EC.3.4.22.32) in the absence and presence of urea, Guanidine hydrochloride and ethanol; four intermediate states could be identified: two activity-enhanced state obtained in the presence of 5 M urea and 2 M GnHCl, termed X and X', respectively, and a third, similarly active state closely resembling the native protein in the presence of 8-9 M urea, termed Y. The enhanced activity of these states is due to local conformational changes accompanied by increased dynamics in the active site. Further, the enzyme does not lose its activity after substantial tertiary structure changes in 8-9 M urea (Y state), suggesting that active site containing domain is more resistant to chemical denaturation than the other structural domain. This makes stem bromelain and in general cysteine proteases an exception to the hypothesis that active site is the most labile part of enzyme.

Anilino Naphthalenesulfonates↗