Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment

Fatih Demir; Elina Kovalenko; Moritz Lassé; Esben B Svenningsen; Jens M Bernth Jensen; Anja M Billing; Kathrin Groeneveld; Arvid Hutzfeldt; Lars Nilges; João P L Guerra; Krzysztof J Pietrzak-Lichwa; Yifan Tan; Elizabeth Colby; Annette G Hansen; Naziia Kurmasheva; David Olagnier; Dongwoo Choi; Mika M Richter; Sandra D Laufer; Fabian Braun; Sally A Johnson; Marcus Krüger; Tobias B Huber; Elion Hoxha; Oliver M Steinmetz; Ralf Mrowka; Simon Melderis; Moin A Saleem; Thomas B Poulsen; Thomas Poulsen; Gregers R Andersen; Steffen Thiel; Anne Troldborg; Markus M Rinschen

doi:10.1038/s44318-025-00598-8

Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment

Fatih Demir
, Elina Kovalenko
, Moritz Lassé
, Esben B Svenningsen
, Jens M Bernth Jensen
, Anja M Billing
, Kathrin Groeneveld
, Arvid Hutzfeldt
, Lars Nilges
, João P L Guerra
, Krzysztof J Pietrzak-Lichwa
, Yifan Tan
, Elizabeth Colby
, Annette G Hansen
, Naziia Kurmasheva
, David Olagnier
, Dongwoo Choi
, Mika M Richter
, Sandra D Laufer
, Fabian Braun

Sally A Johnson, Marcus Krüger, Tobias B Huber, Elion Hoxha, Oliver M Steinmetz, Ralf Mrowka, Simon Melderis, Moin A Saleem, Thomas B Poulsen, Thomas Poulsen, Gregers R Andersen, Steffen Thiel, Anne Troldborg, Markus M Rinschen^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

1 Citation (Scopus)

Abstract

Dysregulated proteolysis is central to autoimmune pathogenesis. The complement cascade, a major protease network, generates fragments that modulate immunity and tissue injury. We developed a scalable blood plasma N-terminomics workflow that markedly expands detection of proteolytic events in vitro and in vivo. Applied to 143 systemic lupus erythematosus (SLE) patients, Multi-Omics Factor Analysis (MOFA) linked N-terminal signatures to immunological and clinical heterogeneity. This revealed a previously unrecognized complement fragment, C3-LHF1, encompassing the C345C domain and rivaling, based on intensity detected by mass spectrometry, the abundance of canonical fragments like C3a and C3b. C3-LHF1 associated with renal function and remission in lupus nephritis, and exhibited dual functions: inhibiting classical and lectin complement pathways and acting as a partial IL6ST (gp130) agonist, independent of IL6Rα. In human kidney organoids, C3-LHF1 induced JAK/STAT3 signaling, amplified TNFα-driven CXCL10 secretion, and reduced podocyte marker expression, suggesting a role in tissue remodeling. These findings reveal unanticipated complexity in complement-mediated signaling and provide a comprehensive atlas of protein N-termini in human plasma, which enables discovery of novel immunoregulatory mechanisms and therapeutic targets in inflammatory disease.

Original language	English
Journal	The EMBO Journal
Pages (from-to)	7721-7758
Number of pages	38
ISSN	0261-4189
DOIs	https://doi.org/10.1038/s44318-025-00598-8
Publication status	Published - 15 Dec 2025

Keywords

LHF
N-terminomics
Proteolysis
SHUNTER
SLE

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Demir, F., Kovalenko, E., Lassé, M., Svenningsen, E. B., Bernth Jensen, J. M., Billing, A. M., Groeneveld, K., Hutzfeldt, A., Nilges, L., Guerra, J. P. L., Pietrzak-Lichwa, K. J., Tan, Y., Colby, E., Hansen, A. G., Kurmasheva, N., Olagnier, D., Choi, D., Richter, M. M., Laufer, S. D., ... Rinschen, M. M. (2025). Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment. The EMBO Journal, 7721-7758. https://doi.org/10.1038/s44318-025-00598-8

@article{6a648e374d1d4a4d8adc298bc1a0a82f,

title = "Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment",

abstract = "Dysregulated proteolysis is central to autoimmune pathogenesis. The complement cascade, a major protease network, generates fragments that modulate immunity and tissue injury. We developed a scalable blood plasma N-terminomics workflow that markedly expands detection of proteolytic events in vitro and in vivo. Applied to 143 systemic lupus erythematosus (SLE) patients, Multi-Omics Factor Analysis (MOFA) linked N-terminal signatures to immunological and clinical heterogeneity. This revealed a previously unrecognized complement fragment, C3-LHF1, encompassing the C345C domain and rivaling, based on intensity detected by mass spectrometry, the abundance of canonical fragments like C3a and C3b. C3-LHF1 associated with renal function and remission in lupus nephritis, and exhibited dual functions: inhibiting classical and lectin complement pathways and acting as a partial IL6ST (gp130) agonist, independent of IL6Rα. In human kidney organoids, C3-LHF1 induced JAK/STAT3 signaling, amplified TNFα-driven CXCL10 secretion, and reduced podocyte marker expression, suggesting a role in tissue remodeling. These findings reveal unanticipated complexity in complement-mediated signaling and provide a comprehensive atlas of protein N-termini in human plasma, which enables discovery of novel immunoregulatory mechanisms and therapeutic targets in inflammatory disease.",

keywords = "LHF, N-terminomics, Proteolysis, SHUNTER, SLE",

author = "Fatih Demir and Elina Kovalenko and Moritz Lass{\'e} and Svenningsen, \{Esben B\} and \{Bernth Jensen\}, \{Jens M\} and Billing, \{Anja M\} and Kathrin Groeneveld and Arvid Hutzfeldt and Lars Nilges and Guerra, \{Jo{\~a}o P L\} and Pietrzak-Lichwa, \{Krzysztof J\} and Yifan Tan and Elizabeth Colby and Hansen, \{Annette G\} and Naziia Kurmasheva and David Olagnier and Dongwoo Choi and Richter, \{Mika M\} and Laufer, \{Sandra D\} and Fabian Braun and Johnson, \{Sally A\} and Marcus Kr{\"u}ger and Huber, \{Tobias B\} and Elion Hoxha and Steinmetz, \{Oliver M\} and Ralf Mrowka and Simon Melderis and Saleem, \{Moin A\} and Poulsen, \{Thomas B\} and Thomas Poulsen and Andersen, \{Gregers R\} and Steffen Thiel and Anne Troldborg and Rinschen, \{Markus M\}",

note = "{\textcopyright} 2025. The Author(s).",

year = "2025",

month = dec,

day = "15",

doi = "10.1038/s44318-025-00598-8",

language = "English",

pages = "7721--7758",

journal = "The EMBO Journal",

issn = "0261-4189",

publisher = "Springer Science and Business Media Deutschland GmbH",

}

Demir, F , Kovalenko, E, Lassé, M, Svenningsen, EB, Bernth Jensen, JM , Billing, AM, Groeneveld, K, Hutzfeldt, A, Nilges, L, Guerra, JPL , Pietrzak-Lichwa, KJ, Tan, Y, Colby, E, Hansen, AG, Kurmasheva, N , Olagnier, D , Choi, D, Richter, MM, Laufer, SD, Braun, F, Johnson, SA, Krüger, M, Huber, TB, Hoxha, E, Steinmetz, OM, Mrowka, R, Melderis, S, Saleem, MA, Poulsen, TB, Poulsen, T , Andersen, GR , Thiel, S , Troldborg, A & Rinschen, MM 2025, 'Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment', The EMBO Journal, pp. 7721-7758. https://doi.org/10.1038/s44318-025-00598-8

TY - JOUR

T1 - Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment

AU - Demir, Fatih

AU - Kovalenko, Elina

AU - Lassé, Moritz

AU - Svenningsen, Esben B

AU - Bernth Jensen, Jens M

AU - Billing, Anja M

AU - Groeneveld, Kathrin

AU - Hutzfeldt, Arvid

AU - Nilges, Lars

AU - Guerra, João P L

AU - Pietrzak-Lichwa, Krzysztof J

AU - Tan, Yifan

AU - Colby, Elizabeth

AU - Hansen, Annette G

AU - Kurmasheva, Naziia

AU - Olagnier, David

AU - Choi, Dongwoo

AU - Richter, Mika M

AU - Laufer, Sandra D

AU - Braun, Fabian

AU - Johnson, Sally A

AU - Krüger, Marcus

AU - Huber, Tobias B

AU - Hoxha, Elion

AU - Steinmetz, Oliver M

AU - Mrowka, Ralf

AU - Melderis, Simon

AU - Saleem, Moin A

AU - Poulsen, Thomas B

AU - Poulsen, Thomas

AU - Andersen, Gregers R

AU - Thiel, Steffen

AU - Troldborg, Anne

AU - Rinschen, Markus M

PY - 2025/12/15

Y1 - 2025/12/15

N2 - Dysregulated proteolysis is central to autoimmune pathogenesis. The complement cascade, a major protease network, generates fragments that modulate immunity and tissue injury. We developed a scalable blood plasma N-terminomics workflow that markedly expands detection of proteolytic events in vitro and in vivo. Applied to 143 systemic lupus erythematosus (SLE) patients, Multi-Omics Factor Analysis (MOFA) linked N-terminal signatures to immunological and clinical heterogeneity. This revealed a previously unrecognized complement fragment, C3-LHF1, encompassing the C345C domain and rivaling, based on intensity detected by mass spectrometry, the abundance of canonical fragments like C3a and C3b. C3-LHF1 associated with renal function and remission in lupus nephritis, and exhibited dual functions: inhibiting classical and lectin complement pathways and acting as a partial IL6ST (gp130) agonist, independent of IL6Rα. In human kidney organoids, C3-LHF1 induced JAK/STAT3 signaling, amplified TNFα-driven CXCL10 secretion, and reduced podocyte marker expression, suggesting a role in tissue remodeling. These findings reveal unanticipated complexity in complement-mediated signaling and provide a comprehensive atlas of protein N-termini in human plasma, which enables discovery of novel immunoregulatory mechanisms and therapeutic targets in inflammatory disease.

AB - Dysregulated proteolysis is central to autoimmune pathogenesis. The complement cascade, a major protease network, generates fragments that modulate immunity and tissue injury. We developed a scalable blood plasma N-terminomics workflow that markedly expands detection of proteolytic events in vitro and in vivo. Applied to 143 systemic lupus erythematosus (SLE) patients, Multi-Omics Factor Analysis (MOFA) linked N-terminal signatures to immunological and clinical heterogeneity. This revealed a previously unrecognized complement fragment, C3-LHF1, encompassing the C345C domain and rivaling, based on intensity detected by mass spectrometry, the abundance of canonical fragments like C3a and C3b. C3-LHF1 associated with renal function and remission in lupus nephritis, and exhibited dual functions: inhibiting classical and lectin complement pathways and acting as a partial IL6ST (gp130) agonist, independent of IL6Rα. In human kidney organoids, C3-LHF1 induced JAK/STAT3 signaling, amplified TNFα-driven CXCL10 secretion, and reduced podocyte marker expression, suggesting a role in tissue remodeling. These findings reveal unanticipated complexity in complement-mediated signaling and provide a comprehensive atlas of protein N-termini in human plasma, which enables discovery of novel immunoregulatory mechanisms and therapeutic targets in inflammatory disease.

KW - LHF

KW - N-terminomics

KW - Proteolysis

KW - SHUNTER

KW - SLE

UR - https://www.scopus.com/pages/publications/105020415009

U2 - 10.1038/s44318-025-00598-8

DO - 10.1038/s44318-025-00598-8

M3 - Journal article

C2 - 41145914

SN - 0261-4189

SP - 7721

EP - 7758

JO - The EMBO Journal

JF - The EMBO Journal

ER -

Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment

Abstract

Keywords

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