Biomedical subjects

Florence Teillet

Publications and source records attributed to Florence Teillet.

2 recordsLinked to original sources

PubMed2005

Functional characterization of complement proteases C1s/mannan-binding lectin-associated serine protease-2 (MASP-2) chimeras reveals the higher C4 recognition efficacy of the MASP-2 complement control protein modules.

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s(MASP-2 CCP1/2) and C1s(MASP-2 SP), respectively) were expressed and characterized chemically and functionally. Whereas C1s(MASP-2 SP) was recovered as an active enzyme, C1s(MASP-2 CCP1/2) was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s(MASP-2 SP) and C1s had Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21-27-fold higher kcat/Km ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s(MASP-2 CCP1/2) retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.

Amino Acid Sequence↗

PubMed2005

The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases.

Mannan-binding lectin (MBL) is an oligomeric C-type lectin assembled from homotrimeric structural units that binds to neutral carbohydrates on microbial surfaces. It forms individual complexes with MBL-associated serine proteases (MASP)-1, -2, -3 and a truncated form of MASP-2 (MAp19) and triggers the lectin pathway of complement through MASP-2 activation. To characterize the oligomerization state of the two major MBL forms present in human serum, both proteins were analyzed by mass spectrometry. Mass values of 228,098 +/- 170 Da (MBL-I) and 304,899 +/- 229 Da (MBL-II) were determined for the native proteins, whereas reduction of both species yielded a single chain with an average mass of 25,340 +/- 18 Da. This demonstrates that MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit. As shown by surface plasmon resonance spectroscopy, trimeric and tetrameric MBL bound to immobilized mannose-BSA and N-acetylglucosamine-BSA with comparable K(D) values (2.2 and 0.55 nM and 1.2 and 0.96 nM, respectively). However, tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for both carbohydrates. In contrast, no significant difference was detected for binding of the recombinant MASPs or MAp19 to immobilized trimeric or tetrameric MBL. As shown by gel filtration, both MBL species formed 1:2 complexes with MASP-3 or MAp19. These results provide the first precise analysis of the major human MBL oligomers. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs.

Acetylglucosamine↗