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 Skjødt, Claus Koch, Peter Garred

Research output: Contribution to journalJournal articleResearchpeer-review

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 6.4mg/l (range: 2-12.9mg/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 languageEnglish
JournalImmunobiology
Volume215
Issue number11
Pages (from-to)921-931
ISSN0171-2985
DOIs
Publication statusPublished - 2010

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Mannose-Binding Protein-Associated Serine Proteases
Serum
CHO Cells

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@article{00bb2500eef011deaefb000ea68e967b,
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 6.4mg/l (range: 2-12.9mg/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 Skj{\o}dt and Claus Koch and Peter Garred",
note = "Udgivelsesdato: 2009-Nov-23",
year = "2010",
doi = "10.1016/j.imbio.2009.10.006",
language = "English",
volume = "215",
pages = "921--931",
journal = "Immunobiology",
issn = "0171-2985",
publisher = "Elsevier",
number = "11",

}

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; Jie Ma, Ying; Weiss, Gudrun; Skjødt, Karsten; Koch, Claus; Garred, Peter.

In: Immunobiology, Vol. 215, No. 11, 2010, p. 921-931.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

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 - Skjødt, Karsten

AU - Koch, Claus

AU - Garred, Peter

N1 - Udgivelsesdato: 2009-Nov-23

PY - 2010

Y1 - 2010

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 6.4mg/l (range: 2-12.9mg/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 6.4mg/l (range: 2-12.9mg/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

VL - 215

SP - 921

EP - 931

JO - Immunobiology

JF - Immunobiology

SN - 0171-2985

IS - 11

ER -