Proteolytic inactivation of nuclear alarmin high-mobility group box 1 by complement protease C1s during apoptosis

Abstract

Effective clearance of apoptotic cells by phagocytes prevents the release of intracellular alarmins and manifestation of autoimmunity. This prompt efferocytosis is complemented by intracellular proteolytic degradation that occurs within the apoptotic cells and in the efferosome of the phagocytes. Although the role of extracellular proteases in apoptotic cells clearance is unknown, the strong association of congenital C1s deficiency with Systemic Lupus Erythematosus highlights the protective nature that this extracellular protease has against autoimmunity. The archetypical role of serine protease C1s as the catalytic arm of C1 complex (C1qC1r2C1s2) involve in the propagation of the classical complement pathway could not provide the biological basis for this association. However, a recent observation of the ability of C1 complex to cleave a spectrum of intracellular cryptic targets exposed during apoptosis provides a valuable insight to the underlying protective mechanism. High-mobility group box 1 (HMGB1), an intracellular alarmin that is capable of inducing the formation of antinuclear autoantibodies and causes lupus-like conditions in mice, is identified as a novel potential target by bioinformatics analysis. This is verified experimentally with C1s, both in its purified and physiological form as C1 complex, cleaving HMGB1 into defined fragments of 19 and 12 kDa. This cleavage diminishes HMGB1 ability to enhance lipopolysaccharide mediated pro-inflammatory cytokines production from monocytes, macrophages and dendritic cells. Further mass spectrometric analysis of the C1 complex treated apoptotic cellular proteins demonstrated additional C1s substrates and revealed the complementary role of C1s in apoptotic cells clearance through the proteolytic cleavage of intracellular alarmins and autoantigens. C1 complex may have evolved as, besides the bacteriolytic arm of antibodies in which it activates the complement cascade, a tissue renewal mechanism that reduces the immunogenicity of apoptotic tissue debris and decreases the likelihood of autoimmunity.

Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation

“…This was consistent with our earlier observation that NCL was cleaved by C1 proteases (48). Four protein spots were identified as NPM1 or its fragments, which was also anticipated (48,54). One Cy2dominant protein of ∼80 kDa (spot 2) was identified as C1s and, based on the size, it corresponded to the proenzyme form of C1s (Table I).…”

“…If nucleoli indeed induce autoantibodies as primary immunogens, it would imply the presence of intrinsic adjuvant elements. In this context, C1 proteases have been found to cleave the nuclear DAMP HMGB1 and inactivate its adjuvant activity (54,66).…”

Cell death and immunity in cancer: From danger signals to mimicry of pathogen defense responses

“…TGFβ, IL10, platelet‐activating factor, and PGE 2 ; or even secretion of soluble immune‐checkpoints like PD‐L1 . Last but not least, tolerogenic apoptosis can also directly inactivate potent DAMPs like HMGB1 via direct degradation through complement protease C1s or via oxidation facilitated by caspases …”

Complement the cell death

“…13 HMGB1 acts as an adjuvant in lipopolysaccharide (LPS) activation of monocytes, macrophages and dendritic cells. It enables low doses of LPS, which is otherwise poorly stimulatory, to activate these cells to produce IL-6 and TNF α .…”

“…HMGB1 contains two cleavage sites for the complement protease C1s and, after C1s cleavage, this adjuvant activity of HMGB1 is diminished as it no longer synergizes with LPS in IL-6 and TNF α induction. 13 This suggests an extracellular mechanism that contributes to the trimming of cellular residues and inactivation of alarmins and probably autoantigens as well (Figure 1). …”