Substitution of Mannan-Binding Lectin (MBL)-Deficient Serum With Recombinant MBL Results in the Formation of New MBL/MBL-Associated Serine Protease Complexes

Abstract

The lectin pathway (LP) of complement activation depends on the activation of the MBL-associated serine proteases (MASPs) circulating in complex with mannan-binding lectin (MBL). MBL deficiency is the most common complement deficiency and has been associated with several pathological conditions. As we had previously shown, plasma-derived MBL (pdMBL) contains pre-activated MASPs that upon in vivo pdMBL substitution results in restoration of MBL concentrations but no LP functionality due to immediate inactivation of pdMBL-MASP complexes upon infusion. In this study, we analyzed MBL-sufficient and -deficient serum by size-exclusion chromatography for complexes of LP activation. In both sera, we identified non-bound free forms of MASP-2 and to lesser extent MASP-1/3. After addition of recombinant MBL (rMBL) to MBL-deficient serum, these free MASPs were much less abundantly present, which is highly suggestive for the formation of high-molecular complexes that could still become activated upon subsequent ligand binding as shown by a restoration of C4-deposition of MBL-deficient serum. Ficolin (FCN)-associated MASPs have been described to redistribute to ligand-bound MBL, hereby forming new MBL/MASP complexes. However, reconstitution of MBL-deficient serum with rMBL did not change the relative size of the FCN molecules suggestive for a limited redistribution in fluid phase of already formed complexes. Our findings demonstrate that rMBL can associate with free non-bound MASPs in fluid phase while preserving full restoration of LP functionality. In contrast to pdMBL products containing pre-activated MASPs which become inactivated almost immediately, these current data provide a rationale for substitution studies using rMBL instead.

Keywords: MBL-associated serine protease; mannan-binding lectin–MBL-associated serine protease complexes; recombinant MBL; redistribution; size-exclusion chromatography.

Figure 1

Direct correlation between elution fraction and size. (A) Elution profile of low-weight and height-weight markers on the Superdex™ 200 10/300 GL in veronal-buffered saline (1 M NaCl, pH 7.4). (B) A strong correlation (r² = 0.994) is found between the elution volume and the marker size.

Figure 2

Size-exclusion chromatography (SEC) of mannan-binding lectin (MBL)-deficient and MBL-sufficient serum. SEC of MBL-deficient serum (A,C) or MBL-sufficient serum (B,D). Serum was fractionated on a Superdex™ 200 10/300 GL column (A,C). Fractions were tested for the presence of MBL, or MBL–MBL-associated serine protease (MASP)-1/3, MBL–MASP-2 complexes on mannan-coated plates and compared with a pool of normal human sera (NHS). (B,D) Mannan-coated plates were pre-incubated with fixed amount of recombinant MBL, and followed by the different fractions of MBL-deficient or MBL-sufficient serum, and the formation of new MBL–MASP-1/3 or MBL–MASP-2 complexes was detected and compared with a pool of NHS.

Figure 3

Size-exclusion chromatography (SEC) of mannan-binding lectin (MBL)-reconstituted MBL-deficient serum. SEC of MBL-deficient serum reconstituted to 50 µg/ml MBL. (A) Fractions were tested for the presence of MBL–MBL-associated serine protease (MASP)-1/3 and MBL–MASP-2 complexes on mannan-coated plates. (B) Mannan-coated plates were pre-incubated with MBL, and followed by the different fractions of reconstituted MBL-deficient serum, and the formation of MBL–MASP-1/3 and MBL–MASP-2 complexes was detected.

Figure 4

Functional binding of ficolin (FCN)-2 and FCN-3. Effect on the distribution of FCN-2 and FCN-3 after reconstitution to 50 µg/ml mannan-binding lectin (MBL). (A) Fractions were tested for the ligand binding of FCN-2 before and after reconstitution. (B) Fractions were tested for the ligand binding of FCN-3 before and after reconstitution.