Inhibition of alternative pathway factor D by factor B ‐related synthetic hexapeptides

Lesavre, Philippe; Guillard, Marie‐Hélène; Halbwachs‐Mecarelli, Lise

doi:10.1002/eji.1830120317

“…Since the thioester substrate was present at subsaturating concentrations, the failure of the peptides to inhibit the enzyme implies that their affinity for binding to the active site of Factor D has a limit of K D g 2 mM. The failure of peptides derived from the cleavage site of Factor B to inhibit the activity of Factor D toward thioester substrates is not consistent with some published reports (24,25). The reason for this discrepancy is not known, though it might be explained if the Factor D used in these published experiments was contaminated with trace amounts of other proteases.…”

Section: Activity Of Factor D Toward Thioester and P-nitroanilidementioning

confidence: 60%

Induced Fit Activation Mechanism of the Exceptionally Specific Serine Protease, Complement Factor D^†

Taylor

¹

,

Bixler

²

,

Budman

³

et al. 1999

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We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM /=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.

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“…The enzyme is also highly specific, as it cleaves its only known substrate, factor B, only when factor B is bound to C3b or C3(H # O) [44]. Several hexapeptides corresponding to the factor B sequence surrounding the bond that is cleaved by factor D were studied [48]. The peptides were assessed for their ability to inhibit factor D enzymic activity and for their susceptibility to cleavage by several SPs including factor D. The peptides were all able to inhibit factor B cleavage by factor D, but were not substrates for factor D. Active-site mapping of factor D with peptide thioesters revealed some interesting features [19,49].…”

Section: Discussionmentioning

confidence: 99%

Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B

WILLIAMS

¹

,

Hinshelwood

²

,

Perkins

³

et al. 1999

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Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys(267)-->Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of (125)I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.

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“…The enzyme is also highly specific, as it cleaves its only known substrate, factor B, only when factor B is bound to C3b or C3(H # O) [44]. Several hexapeptides corresponding to the factor B sequence surrounding the bond that is cleaved by factor D were studied [48]. The peptides were assessed for their ability to inhibit factor D enzymic activity and for their susceptibility to cleavage by several SPs including factor D. The peptides were all able to inhibit factor B cleavage by factor D, but were not substrates for factor D. Active-site mapping of factor D with peptide thioesters revealed some interesting features [19,49].…”

Section: Discussionmentioning

confidence: 99%

Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B

Williams

¹

,

Hinshelwood

²

,

Perkins

³

et al. 1999

Biochemical Journal

View full text Add to dashboard Cite

Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys(267)-->Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of (125)I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.

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Inhibition of alternative pathway factor D by factor B ‐related synthetic hexapeptides

Cited by 12 publications

References 15 publications

Induced Fit Activation Mechanism of the Exceptionally Specific Serine Protease, Complement Factor D^†

Induced Fit Activation Mechanism of the Exceptionally Specific Serine Protease, Complement Factor D^†

Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B

Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B

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Inhibition of alternative pathway factor D by factor B ‐related synthetic hexapeptides

Cited by 12 publications

References 15 publications

Induced Fit Activation Mechanism of the Exceptionally Specific Serine Protease, Complement Factor D†

Induced Fit Activation Mechanism of the Exceptionally Specific Serine Protease, Complement Factor D†

Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B

Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B

Contact Info

Product

Resources

About

Induced Fit Activation Mechanism of the Exceptionally Specific Serine Protease, Complement Factor D^†

Induced Fit Activation Mechanism of the Exceptionally Specific Serine Protease, Complement Factor D^†