Identification of the Site of Human Mannan-Binding Lectin Involved in the Interaction with Its Partner Serine Proteases: The Essential Role of Lys55

Teillet, F; Lacroix, Monique; Thiel, Steffen; Weilguny, Dietmar; Agger, Teit; Arlaud, Gérard J.; Thielens, Nicole M.

doi:10.4049/jimmunol.178.9.5710

Cited by 54 publications

(62 citation statements)

References 38 publications

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“…The rMAP-1 was able to almost completely inhibit the C4 deposition despite the fact that it was added in sequence after MASP-2, thus indicating that it could displace rMASP-2 from rMBL. This is in agreement with results demonstrating that the same amino acid residues in MBL are essential not only for MASP-1 and MASP-3 binding but also MASP-2 binding (28).…”

Section: Discussionsupporting

confidence: 93%

A Novel Mannose-binding Lectin/Ficolin-associated Protein Is Highly Expressed in Heart and Skeletal Muscle Tissues and Inhibits Complement Activation

Skjoedt

Hummelshøj

Palarasah

et al. 2010

Journal of Biological Chemistry

139

146

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The human lectin complement pathway involves circulating complexes consisting of mannose-binding lectin (MBL) or three ficolins (ficolin-1, -2, and -3) in association with three MBL/ ficolin-associated serine proteases (MASP) (MASP-1, -2, and -3) and a nonenzymatic sMAP. MASP-1 and MASP-3 (MASP1 isoforms 1 and 2, respectively) are splice variants of the MASP1 gene, whereas MASP-2 and sMAP are splice variants of the MASP2 gene. We have identified a novel serum protein of 45 kDa that is associated with MBL and the ficolins. This protein is named MBL/ficolin-associated protein 1 (MAP-1 corresponding to MASP1 isoform 3). The transcript generating MAP-1 (MASP1_v3) contains exons 1-8 and a novel exon encoding an in-frame stop codon. The corresponding protein lacks the serine protease domains but contains most of the common heavy chain of MASP-1 and MASP-3. Additionally MAP-1 contains 17 unique C-terminal amino acids. By use of quantitative PCR and MAP-1-specific immunohistochemistry, we found that MAP-1 is highly expressed in myocardial and skeletal muscle tissues as well as in liver hepatocytes with a different expression profile than that observed for MASP-1 and MASP-3. MAP-1 co-precipitated from human serum with MBL, ficolin-2, and ficolin-3, and recombinant MAP-1 was able to inhibit complement C4 deposition via both the ficolin-3 and MBL pathway. In conclusion we have identified a novel 45-kDa serum protein derived from the MASP1 gene, which is highly expressed in striated muscle tissues. It is found in complex with MBL and ficolins and may function as a potent inhibitor of the complement system in vivo.Activation of the complement system is accomplished via three different initiation pathways: the alternative pathway, the classical pathway, and the lectin pathway (1). In humans, four recognition molecules of the lectin pathway have been described: mannose-binding lectin (MBL), 3 ficolin-1 (also called M-ficolin), ficolin-2 (also called L-ficolin), and ficolin-3 (also called H-ficolin or Hakata antigen) (2). MBL and the ficolins bind structures on different classes of microorganisms and are involved in sequestration and removal of dying host cells (2, 3). Three MBL/ficolin-associated serine proteases have been described so far (MASP-1, MASP-2, and MASP-3), as well as a protein lacking a serine protease domain named sMAP or MAp19 (4). Present consensus places MASP-2 as the main initiator of the lectin complement pathway by cleaving C4 and C2 to form the C4b2a complex leading to further downstream complement activation (5). Although the functions of the other MASPs are poorly understood, MASP-1 appears to play a role as an amplifier of complement activation (6, 7). Additionally, MASP-1 is able to cleave fibrinogen to fibrin, whereas MASP-2 is able to generate active thrombin by cleavage of prothrombin (8,9). No conclusive biological function has yet been attributed to MASP-3 and sMAP. MASP-1 and MASP-2 were originally named after their association with MBL (5, 10). Subsequently, MASP-3 and sMAP (also named Map19) we...

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Section: Discussionsupporting

confidence: 93%

A Novel Mannose-binding Lectin/Ficolin-associated Protein Is Highly Expressed in Heart and Skeletal Muscle Tissues and Inhibits Complement Activation

Skjoedt

Hummelshøj

Palarasah

et al. 2010

Journal of Biological Chemistry

139

146

View full text Add to dashboard Cite

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“…All mutants were secreted in the culture medium, with yields of 8 -10 g/ml, similar to those obtained for the wild-type form, except E216A and D226A, which were produced at 1 and 5 g/ml, respectively. All MASP-3 variants were purified by ion-exchange chromatography on a Q-Sepharose Fast Flow column (GE Healthcare) followed by high pressure gel permeation on a TSK G3000 SWG column (Toso Haas), as described previously (25). This latter step allowed us to check that all MASP-3 variants retained the ability to associate as homodimers.…”

Section: Methodsmentioning

confidence: 99%

“…The x-ray structure of the CUB 1 -EGF-CUB 2 segment of rat MASP-2 was also solved, but in contrast no Ca 2ϩ ion could be observed in the CUB modules, and therefore a different model for interaction with MBL was proposed (22). On the other hand, expression of site-directed mutants of human and rat MBL (24,25) and ficolins (26,27) has recently provided evidence that these proteins associate with the MASPs and MAp19 through a major ionic interaction involving a conserved lysine residue.…”

mentioning

confidence: 99%

Crystal Structure of the CUB1-EGF-CUB2 Domain of Human MASP-1/3 and Identification of Its Interaction Sites with Mannan-binding Lectin and Ficolins

Teillet¹,

Gaboriaud

Lacroix³

et al. 2008

Journal of Biological Chemistry

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(CUB 2 ), plus one and two water molecules, respectively. To identify the residues involved in interaction of MASP-1 and -3 with MBL and L-and H-ficolins, 27 point mutants of human MASP-3 were generated, and their binding properties were analyzed using surface plasmon resonance spectroscopy. These mutations map two homologous binding sites contributed by modules CUB 1 and CUB 2 , located in close vicinity of their Ca 2؉ -binding sites and stabilized by the Ca 2؉ ion. This information allows us to propose a model of the MBL-MASP-1/3 interaction, involving a major electrostatic interaction between two acidic Ca 2؉ ligands of MASP-1/3 and a conserved lysine of MBL. Based on these and other data, a schematic model of a MBL⅐MASP complex is proposed.The lectin pathway of complement is increasingly recognized as an important component of innate immunity against pathogens. This pathway is triggered by oligomeric lectins that recognize patterns of neutral and acetylated carbohydrates on the surface of pathogens and share the ability to associate with and trigger activation of modular proteases termed mannanbinding lectin-associated serine proteases (MASPs) 3 (1, 2). Four such oligomeric lectins have been described: mannanbinding lectin (MBL) and ficolins L, H, and M (3-7). These proteins all assemble as oligomers of homotrimeric subunits, each comprising N-terminal collagen-like triple helices prolonged by recognition domains endowed with lectin-like binding activities. There are three different MASPs (MASP-1, -2, and -3) (4,8,9), and these feature modular structures homologous to those of C1r and C1s, the proteases of the C1 complex of complement, with an N-terminal CUB module (10), an epidermal growth factor (EGF)-like module belonging to the Ca 2ϩ -binding subset (11), a second CUB module, two complement control protein (CCP) modules (12), and a chymotrypsin-like serine protease domain. MASP-1 and MASP-3 are alternative splicing products of the MASP1/3 gene and include different serine protease domains but share identical CUB 1 -EGF-CUB 2 -CCP 1 -CCP 2 segments (9). Likewise, alternative splicing of the MASP2 gene generates MBL-associated protein 19 (MAp19), a truncated protein comprising the N-terminal CUB 1 -EGF segment of MASP-2 prolonged by four residues specific to MAp19 (13,14). From a functional standpoint, the ability of MASP-2 to trigger the lectin pathway of complement is clearly established (8). In contrast, whether MASP-1 is involved in this pathway is still a controversial issue (15,16), and the function of MASP-3 remains elusive.Studies on human (17-20) and rat (21, 22) proteins have established that the MASPs and MAp19 each associate as homodimers through their CUB 1 -EGF segment. In turn, the MASPs and MAp19 each form individual Ca 2ϩ -dependent complexes with MBL and the ficolins. The interaction involves a major site located in the CUB 1 -EGF moiety of each protein but is strengthened by module CUB 2 (18,19,22,23). Resolution of the x-ray structure of human MAp19 has allowed identification of a Ca 2ϩ...

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“…Deletion of the CCP modules located at the N-or C-terminal ends (DCCP22-23 and DCCP27-30) had no or little impact on the MBL and L-ficolin binding capacities. In contrast, removal of the central CCP module pairs 24-25 (DCCP22-25) and [25][26] reduced the interaction in a significant manner. Interestingly, the CCP22-25 fragment (DCCP26-30) had a higher binding capacity than the CCP22-26 (DCCP27-30) fragment, suggesting that deletion of Table I.…”

Section: Location Of the Binding Site In Cr1 Ccp22-30mentioning

confidence: 96%

“…Elution from the acetylated BSA-Sepharose column (prepared by coupling 125 mg acetylated BSA to 15 ml CNBractivated Sepharose 4B [GE Healthcare, Vélizy, France]) was carried out using 1 M sodium acetate and 5 mM EDTA (pH 7.4). Recombinant MBL and its K55A and K55E variants, produced in Freestyle 293-F cells (Invitrogen Life Technologies, St. Aubin, France) and purified as previously described (26) were provided by NatImmune (Copenhagen, Denmark). The molar concentrations of dimeric MASP-3, tetrameric MBL, tetrameric L-ficolin, and tetrameric H-ficolin were estimated using M r values of 175,200, 305,400, 406,300, and 396,000 and absorbance coefficients at 280 nm (A 1%, 1 cm ) of 13.5, 7.8, 17.6, and 19.4, respectively (25,27).…”

Section: Proteins and Reagentsmentioning

confidence: 99%

Deciphering Complement Receptor Type 1 Interactions with Recognition Proteins of the Lectin Complement Pathway

Jacquet

Lacroix²,

Ancelet³

et al. 2013

The Journal of Immunology

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Complement receptor type 1 (CR1) is a membrane receptor expressed on a wide range of cells. It is involved in immune complex clearance, phagocytosis, and complement regulation. Its ectodomain is composed of 30 complement control protein (CCP) modules, organized into four long homologous repeats (A–D). In addition to its main ligands C3b and C4b, CR1 was reported to interact with C1q and mannan-binding lectin (MBL) likely through its C-terminal region (CCP22–30). To decipher the interaction of human CR1 with the recognition proteins of the lectin complement pathway, a recombinant fragment encompassing CCP22–30 was expressed in eukaryotic cells, and its interaction with human MBL and ficolins was investigated using surface plasmon resonance spectroscopy. MBL and L-ficolin were shown to interact with immobilized soluble CR1 and CR1 CCP22–30 with apparent dissociation constants in the nanomolar range, indicative of high affinity. The binding site for CR1 was located at or near the MBL-associated serine protease (MASP) binding site in the collagen stalks of MBL and L-ficolin, as shown by competition experiments with MASP-3. Accordingly, the mutation of an MBL conserved lysine residue essential for MASP binding (K55) abolished binding to soluble CR1 and CCP22–30. The CR1 binding site for MBL/ficolins was mapped to CCP24–25 of long homologous repeat D using deletion mutants. In conclusion, we show that ficolins are new CR1 ligands and propose that MBL/L-ficolin binding involves major ionic interactions between conserved lysine residues of their collagen stalks and surface exposed acidic residues located in CR1 CCP24 and/or CCP25.

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Identification of the Site of Human Mannan-Binding Lectin Involved in the Interaction with Its Partner Serine Proteases: The Essential Role of Lys55

Cited by 54 publications

References 38 publications

A Novel Mannose-binding Lectin/Ficolin-associated Protein Is Highly Expressed in Heart and Skeletal Muscle Tissues and Inhibits Complement Activation

A Novel Mannose-binding Lectin/Ficolin-associated Protein Is Highly Expressed in Heart and Skeletal Muscle Tissues and Inhibits Complement Activation

Crystal Structure of the CUB1-EGF-CUB2 Domain of Human MASP-1/3 and Identification of Its Interaction Sites with Mannan-binding Lectin and Ficolins

Deciphering Complement Receptor Type 1 Interactions with Recognition Proteins of the Lectin Complement Pathway

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