Complement C7 and clusterin form a complex in circulation, 2024, Massri et al.

[SNT Gatchaman]

Complement C7 and clusterin form a complex in circulation
Massri, Mariam; Toonen, Erik J.M.; Sarg, Bettina; Kremser, Leopold; Grasse, Marco; Fleischer, Verena; Torres-Quesada, Omar; Hengst, Ludger; Skjoedt, Mikkel-Ole; Bayarri-Olmos, Rafael; Rosbjerg, Anne; Garred, Peter; Orth-Höller, Dorothea; Prohászka, Zoltán; Würzner, Reinhard

The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is complement C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated.

To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. A series of proteolytic analyses of serum-purified C7, encompassing Western blot and mass spectrometry analysis, showcased C7 in association with clusterin, an inhibitory complement regulator.

The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Clusterin dissociates the MAC structure by binding to polymerized C9, nevertheless, clusterin binding to the native form of terminal complement proteins has not yet been showcased in vivo. Further assessment of the relationship between C7 and clusterin was assessed via an established enzyme-linked immunosorbent assay (ELISA), in which a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, as well as in serum fractionated by size exclusion chromatography, further verifying the presence of this protein complex in circulation.

The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.

Link | PDF (Frontiers in Immunology)

 

[SNT Gatchaman]

Selected quotes from discussion —

To date, there are a few studies that focus on dissecting the properties of C7 beyond its integral role in MAC assembly.

Our Western blot analysis of serum-purified C7 revealed an additional form of C7 with a molecular weight (75 kDa) lower than that of native C7 (100 kDa). Initially, we believed that a truncated C7 variant, due to alternative splicing, existed in circulation. However, given that we detected C-terminal residues in the 75 kDa band, we excluded the presence of a functionally truncated variant, as truncated proteins do not comprise end terminal sequences. Further analysis of the 75 kDa band by mass spectrometry revealed an abundance of clusterin, and a strong association between C7 and clusterin was identified.

Clusterin (also known as apolipoprotein J) is a ubiquitously expressed apolipoprotein, with various paradoxical functions. Several isoforms are generated from the clusterin protein precursor, the largest being a glycosylated form of a-b heterodimers (75-80 kDa) that is secreted from cells. Clusterin is the first molecule identified to possess chaperone activities outside the cell. It is involved in the clearance of misfolded proteins from the extracellular matrix by binding to pathogenic protein aggregates, inducing their internalization and degradation.

Clusterin inhibits the terminal complement pathway by binding C5b-9, resulting in a lytically inactive complex.

Here, we provided evidence of a complex between clusterin and a terminal complement protein, C7, in circulation.

The C7 gene contains numerous single nucleotide variants, some of which are rare within a given population.

Size exclusion chromatography is a standard method used to characterize protein complexes in medical fluids, without the need to manipulate the environment, such as in the case of capture antibodies. SEC analysis therefore was used to validate the presence of the C7-CLU complex in circulation, revealing that the molecular weight of the complex was much higher than the expected range of 200 kDa. It is important to highlight that the majority of circulatory C7 is not bound to clusterin, as the concentration of the C7-CLU complex was a fraction of serum concentrations of native C7.

The majority of complement proteins are produced in the liver, nevertheless, the liver is not the primary site for C7 synthesis. Studies revealed that C7 is mainly produced in granulocytes, endothelial and bone-marrow-derived cells. [...] If insufficient levels of C7 are present, it acts as a limiting factor, preventing further activation, irrespective of the presence of other inhibitors.

C7 has been reported to augment the activation of plasminogen and protect it from being degraded by a2 antiplasmin.

The regulatory functions of C7 are not well understood yet, nevertheless, both C7 and clusterin have been shown to possess complement regulatory properties. This shared role could provide insight for the association between these two proteins.

We believe that clusterin’s ability to scavenge complement proteins plays a key role in the formation of the C7-CLU complex.

Clusterin expression was reported in different types of endothelial-derived cells and has been shown to play a protective role in endothelial cell function.

 

[SNT Gatchaman]

We have a tag for clusterin.

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