Two Oligomeric Forms of Plasma Ficolin Have Differential Lectin Activity

Abstract

Ficolins are plasma proteins with binding activity for carbohydrates, elastin, and corticosteroids. The ficolin polypeptide has a collagen-like domain that presumably brings three subunits together in a triple helical rod, a C-terminal fibrinogen-like domain (fbg) similar to that of tenascin, which presumably has the binding activities, and a small N-terminal domain that we find to be the primary site for forming the ficolin oligomer. By sedimentation equilibrium we determined that the main plasma form, which we call big ficolin, had mass of 827,000 Da, consistent with 24 subunits. Little ficolin, about half this size, was obtained after binding to a GlcNAc affinity column. Electron microscopy of little ficolin showed a parachute-like structure, with a small globe at one end, corresponding to the 12 N-terminal domains, and the fbg domains clustered together at the ends of the collagen rods. Big ficolin was formed by the face to face fusion of the fbg domains of two little ficolins, leaving the rods and N-terminal domains projecting at opposite ends. Little ficolin maintained a high affinity for the GlcNAc column, and big ficolin had a low affinity or none. The binding sites for ligands may be obscured in this big ficolin oligomer, providing a regulation of their activity.Ficolin was originally isolated as a protein from pig uterus membrane extracts that bound transforming growth factor-␤ (1). Two cDNA clones with very similar sequences, ficolin-␣ and -␤, were obtained by screening a pig uterus cDNA library (2). More recently, ficolins have been identified from human blood as a corticosteroid binding protein, termed hucolin (3), an elastin binding protein, termed EBP-37 (4), and a GlcNAc binding lectin, termed P35 (5). Ficolin cDNAs, termed human ficolin (6), ficolin-1 (7), and P35-related gene (8), were also cloned. cDNA sequences (some of them are partial) suggest that there are two human ficolin genes. One gene codes for human ficolin, ficolin-1, and P35-related gene (which are different names given to this gene product), and the other codes for P35, EBP-37, and hucolin. It is not yet clear how these relate to the two pig genes for ␣ and ␤ ficolins.The amino acid sequence showed that ficolins consist of a short N-terminal domain, a middle collagen-like domain, and a C-terminal fibrinogen-like (fbg) 1 domain (2, 5-7). A similar three-domain structure applies to complement protein C1q and the collectins, which include mannose binding protein, conglutinin, and pulmonary surfactant proteins A and D (SP-A and SP-D) (9 -12). These proteins differ from ficolin in that their C-terminal domain is a C-type lectin instead of fbg, but they all have a middle collagen domain and a small N-terminal domain. The collagen domains assemble these proteins into trimers, and electron microscopy shows that four or six trimers are connected together by the N-terminal domain, leaving the Cterminal lectin domains to project in a multimeric array (13-17).Complement proteins like C1q and collectins play roles in immune defense. Collectin...

Trivalent Recognition Unit of Innate Immunity System

“…Trimeric Formation of FD1-The trimerization of ficolins is thought to be mediated by the N-terminal collagen-like region (13,14). However, we found that the recombinant FD1, which lacks the collagen-like region, forms a trimer in solution as judged by a dynamic light scattering experiment in 0.1-1 mg ml Ϫ1 FD1 solution (apparent molecular mass 86.3-88.9 kDa, data not shown).…”

“…The CRD on MBL, surfactant protein A, and surfactant protein D forms a trimeric structure through a triple ␣-helical coiled-coil at a short neck region between the collagen-like domain and the CRD (10 -12). Ficolins also form trimers (8,13,14), although the mechanism of trimerization is unclear. Both ficolins and collectins form trimer-based multimers that are N-terminally linked by disulfide bonds (15).…”

The Disulfide Bonding Pattern in Ficolin Multimers

“…This structure is also similar to complement protein C1q (21). The primary difference between ficolins and collectins is that the C-terminal globular domain of ficolin is a fibrinogen-like (fbg) domain, whereas that of collectins is a carbohydrate recognition domain (12)(13)(14)26). In addition, collectin family proteins have a short ␣-helical neck region between the collagen and C-terminal domains, whereas ficolins lack this region.…”

“…Both families of proteins form trimer-based multimers; CL-43 and MBP-C form trimers as the native form, whereas ficolins, conglutinin, MBP-A, and SP-D form tetratrimers (12-mers), and SP-A and C1q form hexatrimers (18-mers) (19 -26, 31, 32). In some cases, larger multimers have been found for ficolin (26), MBP (25), and SP-D (19). Collectin trimers are linked to each other by disulfide bonds in the N-terminal domain (32)(33)(34).…”

“…However, some mutants did not bind to the GlcNAc column. These inactive mutants were purified with an antibody affinity column as reported previously (26). SDS-PAGE was carried out using standard techniques.…”