Novel complement regulatory mechanisms in disease

The complement system is one of the most important defence mechanisms against bacteria and pathogens. It acts within the framework of both innate and adaptive immunity. In addition to direct elimination of pathogens, complement also supports waste removal (dying cells, immune complexes, and misfolded proteins) and guides effector mechanisms of the adaptive immune response. Key events in complement activation are the formation of the C3 and C5 convertase enzyme complexes, the release of chemoattractants, the opsonization with C3b, and cell lysis due to the assembly of the membrane-attack complex (MAC). The proteolytic cascade of the complement system must be tightly regulated, since both excessive and insufficient activation significantly contributes to the pathology of many diseases. To protect its own cells and tissues, our body expresses several soluble and membrane-bound complement regulators. Here, we study the function of three of these regulators. Sushi Domain-Containing Protein 4 (SUSD4) is a poorly studied human protein. We show that SUSD4 is a novel complement inhibitor that interferes with the formation of the C3 converts. Additionally, we found that SUSD4 is expressed by breast cancer cells, and that this expression is correlated with a better patient prognosis. Factor I is a well-known complement inhibitor involved in the degradation of the activation products C4b and C3b. We detected factor I expression in breast cancer, and determined that breast cancer cells are able to produce the proteolytically active form of this protein. Analysis of patient data revealed that factor I expression correlates with poor survival rates. Cartilage Oligomeric Matrix Protein (COMP) is a large protein involved in the organisation of collagen in the extracellular matrix. We have previously shown that it can both activate and inhibit the complement system. Here, we determined that COMP expression is unregulated in breast cancer tissue where it contributes to a more aggressive form of cancer. COMP expression correlates with poor prognosis and faster recurrence of the disease. Tumors expressing COMP, grown in vivo, were significantly larger and more invasive as compared to control tumors. Interestingly, COMP affected the metabolism and protein processing machinery of the cancer cells, helping them adapt better to a harsh environment. In summary, this thesis describes novel, disease-related, functions of three complement regulators

Functional analyses of complement convertases using C3 and C5-depleted sera.

C3 and C5 convertases are central stages of the complement cascade since they converge the different initiation pathways, augment complement activation by an amplification loop and lead to a common terminal pathway resulting in the formation of the membrane attack complex. Several complement inhibitors attenuate convertase formation and/or accelerate dissociation of convertase complexes. Functional assays used to study these processes are often performed using purified complement components, from which enzymatic complexes are reconstituted on the surface of erythrocytes or artificial matrices. This strategy enables identification of individual interactions between convertase components and putative regulators but carries an inherent risk of detecting non-physiological interactions that would not occur in a milieu of whole serum. Here we describe a novel, alternative method based on C3 or C5-depleted sera, which support activation of the complement cascade up to the desired stages of convertases. This approach allows fast and simple assessment of the influence of putative regulators on convertase formation and stability. As an example of practical utility of the assay, we performed studies on thioredoxin-1 in order to clarify the mechanism of its influence on complement convertases

Sushi domain-containing protein 4 (SUSD4) inhibits complement by disrupting the formation of the classical C3 convertase.

Recently discovered Sushi domain-containing protein 4 (SUSD4) contains several Sushi or complement control protein domains; therefore, we hypothesized that it may act as complement inhibitor. Two isoforms of human SUSD4, fused to the Fc part of human IgG, were recombinantly expressed in Chinese hamster ovary (CHO) cells. The secreted soluble isoform of SUSD4 (SUSD4b) inhibited the classical and lectin complement pathways by 50% at a concentration of 0.5 μM. This effect was due to the fact that 1 μM SUSD4b inhibited the formation of the classical C3 convertase by 90%. The membrane-bound isoform (SUSD4a) inhibited the classical and alternative complement pathways when expressed on the surface of CHO cells but not when expressed as a soluble, truncated protein. In all functional studies, we used known complement inhibitors as positive controls, while Coxsackie adenovirus receptor, which has no effect on complement, expressed with Fc tag, was a negative control. We also studied the mRNA expression of both isoforms of SUSD4 in a panel of human tissues using quantitative PCR and primarily found SUSD4a in esophagus and brain, while SUSD4b was highly expressed in esophagus, ovary, and heart. Overall, our results show that SUSD4 is a novel complement inhibitor with restricted expression.-Holmquist, E., Okroj, M., Nodin, B., Jirström, K. Blom, A. M. Sushi domain-containing protein 4 (SUSD4) inhibits complement by disrupting the formation of the classical C3 convertase

Validation of alternative convertases assays with acknowledged complement inhibitors.

<p>Alternative convertases were formed on the surface of rE with 5% of C5-depleted serum for 20 minutes in the presence (A) or absence (B) of complement inhibitors or control proteins. Then complement mediated lysis was developed with guinea-pig serum in buffer containing EDTA (A) or subjected to another incubation, where dissociation of the convertases was allowed for additional 5 minutes, followed by washing and addition of guinea-pig serum/EDTA (B). Values were collected from three independent experiments and related to control readout (no protein added to C3/C5 -depleted serum). Symbols *, ** and *** stand for p<0.05, p<0.01 or p<0.001, respectively according to one-way ANOVA.</p

Expected effect on lytic activity of complement inhibitors validated in the assays.

*<p>the noticeable effect is due to inhibition of the amplification loop supported by the alternative complement pathway,</p>**<p>see insert in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047245#pone-0047245-g005" target="_blank">Fig. 5c</a>.</p

Testing of functional depletion of C3 or C5 from serum.

<p>EA were diluted in DGVB⁺⁺ buffer and incubated with 0.5% of C3 or C5 depleted serum supplemented or not with purified C3 or C5, respectively. Results are collected from three independent experiments.</p

Tmax of alternative convertases.

<p>rE were incubated with the C5-depleted serum for indicated time periods and thereafter complement mediated lysis was developed with guinea-pig serum in buffer containing EDTA (A). Values were collected from three independent experiments and related to full lysis of EA. In panel B, rE were incubated with a mix of function blocking antibodies against soluble complement inhibitors FH, C4BP and FI or control antibody against protein S. Symbols * and *** stands for p<0.05 or p<0.001, according to two-way ANOVA. Panel C shows the effect of individual function-blocking antibody measured at 60 minutes. Symbol *** stands for p<0.001 according to one-way ANOVA.</p

Concentration of soluble complement inhibitors in depleted sera (µg/ml ± standard deviation).

<p>Reference values in serum, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047245#pone.0047245-Volankis1" target="_blank">[40]</a>: FI: 35 µg/ml, C4BP: 250 µg/ml, FH: 500 µg/ml.</p