The Disulfide Bonding Pattern in Ficolin Multimers

Abstract

Ficolin is a plasma lectin, consisting of a short Nterminal multimerization domain, a middle collagen domain, and a C-terminal fibrinogen-like domain. The collagen domains assemble the subunits into trimers, and the N-terminal domain assembles four trimers into 12-mers. Two cysteine residues in the N-terminal domain are thought to mediate multimerization by disulfide bonding. We have generated three mutants of ficolin ␣ in which the N-terminal cysteines were substituted by serines (Cys 4 , Cys 24 , and Cys 4 /Cys 24 ). The N-terminal cysteine mutants were produced in a mammalian cell expression system, purified by affinity chromatography, and analyzed under nondenaturing conditions to resolve the multimer structure of the native protein and under denaturing conditions to resolve the disulfidelinked structure. Glycerol gradient sedimentation and electron microscopy in nondenaturing conditions showed that plasma and recombinant wild-type protein formed 12-mers.

Residue Lys57 in the Collagen-Like Region of Human L-Ficolin and Its Counterpart Lys47 in H-Ficolin Play a Key Role in the Interaction with the Mannan-Binding Lectin-Associated Serine Proteases and the Collectin Receptor Calreticulin

“…3, a-c) and top views (d and e). The former showed similar parachute-like structures for both ficolins with, in most cases, four heads connected by a rod-like structure to a small globe at the opposite end, comparable to those described previously for mouse plasma ficolin (44), porcine plasma, and recombinant ␣-ficolins (3,45). In contrast, the top views shown in d and e of Fig.…”

“…Ficolins are assembled from homotrimeric subunits comprising a collagen-like triple helix and a lectin-like domain composed of three fibrinogenlike (FBG) 3 domains. Two cysteines at the N-terminal end of the polypeptide chains form interchain disulfide bonds that mediate assembly into higher oligomerization structures (3,4). In humans, L-and H-ficolins have been characterized in serum whereas Mficolin is mainly expressed by the monocytic cell lineage (5)(6)(7).…”

Trimeric structure and conformational equilibrium of M-ficolin fibrinogen-like domain

“…Ficolins, which are composed of a collagen-like domain at the N-terminus and a fibrinogen-like domain (FBG) at the C-terminus, are one of the most important groups of pattern-recognition molecules in innate immune systems. Ficolins form trimer-based multimers that are mutually linked by intermolecular disulfide bonds at the N-terminal domain (Ohashi & Erickson, 2004). Three human ficolins, M-ficolin in cells and L-ficolin and H-ficolin in serum, have been characterized.…”

Extremes of l-ficolin concentration in children with recurrent infections are associated with single nucleotide polymorphisms in the FCN2 gene

“…At the C-terminus, unlike collectins, a fibrinogen-like domain (FBG) is present, which binds target structures in a lectin-like manner. l-ficolin molecules may also dimerize further (to 24-mers) via FBG regions (probably a regulatory mechanism arising from blocking of sugar-binding sites) [1,2,8].…”