MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

Mikkel-Ole Skjoedt; Yaseelan Palarasah; Lea Munthe-Fog; Ying Jie Ma; Gudrun Weiss; Karsten Skjodt; Claus Koch; Peter Garred; Mikkel-Ole Skjoedt; Yaseelan Palarasah; Lea Munthe-Fog; Ying Jie Ma; Gudrun Weiss; Karsten Skjodt; Claus Koch; Peter Garred

doi:10.1016/j.imbio.2009.10.006

MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

Mikkel-Ole Skjoedt, Yaseelan Palarasah, Lea Munthe-Fog, Ying Jie Ma, Gudrun Weiss, Karsten Skjodt, Claus Koch, Peter Garred, Mikkel-Ole Skjoedt, Yaseelan Palarasah, Lea Munthe-Fog, Ying Jie Ma, Gudrun Weiss, Karsten Skjodt, Claus Koch, Peter Garred

41 Citations (Scopus)

Abstract

Background: The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing and little is known about its biological characteristics. For this reason we expressed recombinant MASP-3 and generated specific monoclonal antibodies to establish biochemical characteristics and to determine the serum levels, the interactions with the LCP recognition molecules and the influence on complement activation of MASP-3. Methods: We expressed rMASP-3 in CHO-DG44 cells and used SDS-PAGE and Western blotting for biochemical characterization. We generated monoclonal antibodies against MASP-3 and developed a quantitative MASP-3 assay to establish the serum levels in 100 Danish blood donors. In addition we assessed the association levels between MASP-3 and Ficolin-2, -3 and MBL using both ELISA and immunoprecipitation techniques. Moreover, we assessed the influence on complement factor C4 deposition. Results: We found the mean serum MASP-3 concentration to be 6.4. mg/l (range: 2-12.9. mg/l) and that MASP-3 in serum is primarily found in complex with Ficolin-3. In contrast to this the MASP-3 association with Ficolin-2 and especially with MBL seems to be less evident. rMASP-3 significantly inhibited Ficolin-3 mediated C4 deposition, while the opposite was the case for rMASP-1. Conclusion: Our results show that MASP-3 is present in relatively high serum concentrations. Moreover, Ficolin-3 is the primary acceptor molecule of MASP-3 among the LCP activator molecules, but MASP-3 appears to down-regulate Ficolin-3 mediated complement activation through the lectin pathway.

Original language	English
Journal	Immunobiology
Volume	215
Issue number	11
Pages (from-to)	921-31
Number of pages	11
ISSN	0171-2985
DOIs	https://doi.org/10.1016/j.imbio.2009.10.006
Publication status	Published - 1 Nov 2010

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Skjoedt, M-O., Palarasah, Y., Munthe-Fog, L., Jie Ma, Y., Weiss, G., Skjodt, K., Koch, C., Garred, P., Skjoedt, M-O., Palarasah, Y., Munthe-Fog, L., Jie Ma, Y., Weiss, G., Skjodt, K., Koch, C., & Garred, P. (2010). MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation. Immunobiology, 215(11), 921-31. https://doi.org/10.1016/j.imbio.2009.10.006

MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation. / Skjoedt, Mikkel-Ole; Palarasah, Yaseelan; Munthe-Fog, Lea et al.

In: Immunobiology, Vol. 215, No. 11, 01.11.2010, p. 921-31.

Research output: Contribution to journal › Journal article › Research › peer-review

Skjoedt, M-O, Palarasah, Y, Munthe-Fog, L, Jie Ma, Y, Weiss, G, Skjodt, K, Koch, C, Garred, P, Skjoedt, M-O, Palarasah, Y, Munthe-Fog, L, Jie Ma, Y, Weiss, G, Skjodt, K, Koch, C & Garred, P 2010, 'MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation', Immunobiology, vol. 215, no. 11, pp. 921-31. https://doi.org/10.1016/j.imbio.2009.10.006

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title = "MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation",

abstract = "Background: The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing and little is known about its biological characteristics. For this reason we expressed recombinant MASP-3 and generated specific monoclonal antibodies to establish biochemical characteristics and to determine the serum levels, the interactions with the LCP recognition molecules and the influence on complement activation of MASP-3. Methods: We expressed rMASP-3 in CHO-DG44 cells and used SDS-PAGE and Western blotting for biochemical characterization. We generated monoclonal antibodies against MASP-3 and developed a quantitative MASP-3 assay to establish the serum levels in 100 Danish blood donors. In addition we assessed the association levels between MASP-3 and Ficolin-2, -3 and MBL using both ELISA and immunoprecipitation techniques. Moreover, we assessed the influence on complement factor C4 deposition. Results: We found the mean serum MASP-3 concentration to be 6.4. mg/l (range: 2-12.9. mg/l) and that MASP-3 in serum is primarily found in complex with Ficolin-3. In contrast to this the MASP-3 association with Ficolin-2 and especially with MBL seems to be less evident. rMASP-3 significantly inhibited Ficolin-3 mediated C4 deposition, while the opposite was the case for rMASP-1. Conclusion: Our results show that MASP-3 is present in relatively high serum concentrations. Moreover, Ficolin-3 is the primary acceptor molecule of MASP-3 among the LCP activator molecules, but MASP-3 appears to down-regulate Ficolin-3 mediated complement activation through the lectin pathway.",

author = "Mikkel-Ole Skjoedt and Yaseelan Palarasah and Lea Munthe-Fog and {Jie Ma}, Ying and Gudrun Weiss and Karsten Skjodt and Claus Koch and Peter Garred and Mikkel-Ole Skjoedt and Yaseelan Palarasah and Lea Munthe-Fog and {Jie Ma}, Ying and Gudrun Weiss and Karsten Skjodt and Claus Koch and Peter Garred",

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doi = "10.1016/j.imbio.2009.10.006",

language = "English",

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T1 - MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

AU - Skjoedt, Mikkel-Ole

AU - Palarasah, Yaseelan

AU - Munthe-Fog, Lea

AU - Jie Ma, Ying

AU - Weiss, Gudrun

AU - Skjodt, Karsten

AU - Koch, Claus

AU - Garred, Peter

AU - Skjoedt, Mikkel-Ole

AU - Palarasah, Yaseelan

AU - Munthe-Fog, Lea

AU - Jie Ma, Ying

AU - Weiss, Gudrun

AU - Skjodt, Karsten

AU - Koch, Claus

AU - Garred, Peter

PY - 2010/11/1

Y1 - 2010/11/1

N2 - Background: The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing and little is known about its biological characteristics. For this reason we expressed recombinant MASP-3 and generated specific monoclonal antibodies to establish biochemical characteristics and to determine the serum levels, the interactions with the LCP recognition molecules and the influence on complement activation of MASP-3. Methods: We expressed rMASP-3 in CHO-DG44 cells and used SDS-PAGE and Western blotting for biochemical characterization. We generated monoclonal antibodies against MASP-3 and developed a quantitative MASP-3 assay to establish the serum levels in 100 Danish blood donors. In addition we assessed the association levels between MASP-3 and Ficolin-2, -3 and MBL using both ELISA and immunoprecipitation techniques. Moreover, we assessed the influence on complement factor C4 deposition. Results: We found the mean serum MASP-3 concentration to be 6.4. mg/l (range: 2-12.9. mg/l) and that MASP-3 in serum is primarily found in complex with Ficolin-3. In contrast to this the MASP-3 association with Ficolin-2 and especially with MBL seems to be less evident. rMASP-3 significantly inhibited Ficolin-3 mediated C4 deposition, while the opposite was the case for rMASP-1. Conclusion: Our results show that MASP-3 is present in relatively high serum concentrations. Moreover, Ficolin-3 is the primary acceptor molecule of MASP-3 among the LCP activator molecules, but MASP-3 appears to down-regulate Ficolin-3 mediated complement activation through the lectin pathway.

AB - Background: The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing and little is known about its biological characteristics. For this reason we expressed recombinant MASP-3 and generated specific monoclonal antibodies to establish biochemical characteristics and to determine the serum levels, the interactions with the LCP recognition molecules and the influence on complement activation of MASP-3. Methods: We expressed rMASP-3 in CHO-DG44 cells and used SDS-PAGE and Western blotting for biochemical characterization. We generated monoclonal antibodies against MASP-3 and developed a quantitative MASP-3 assay to establish the serum levels in 100 Danish blood donors. In addition we assessed the association levels between MASP-3 and Ficolin-2, -3 and MBL using both ELISA and immunoprecipitation techniques. Moreover, we assessed the influence on complement factor C4 deposition. Results: We found the mean serum MASP-3 concentration to be 6.4. mg/l (range: 2-12.9. mg/l) and that MASP-3 in serum is primarily found in complex with Ficolin-3. In contrast to this the MASP-3 association with Ficolin-2 and especially with MBL seems to be less evident. rMASP-3 significantly inhibited Ficolin-3 mediated C4 deposition, while the opposite was the case for rMASP-1. Conclusion: Our results show that MASP-3 is present in relatively high serum concentrations. Moreover, Ficolin-3 is the primary acceptor molecule of MASP-3 among the LCP activator molecules, but MASP-3 appears to down-regulate Ficolin-3 mediated complement activation through the lectin pathway.

U2 - 10.1016/j.imbio.2009.10.006

DO - 10.1016/j.imbio.2009.10.006

M3 - Journal article

C2 - 19939495

SN - 0171-2985

VL - 215

SP - 921

EP - 931

JO - Immunobiology

JF - Immunobiology

IS - 11

ER -

MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

Abstract

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