Award Number : W 81 XWH - 11 - 1 - 0309 TITLE : Targeting the Human Complement Membrane Attack Complex to Selectively Kill Prostate Cancer Cells

Prostate-specific antigen (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high and low grade prostate cancer (PCa) throughout the course of disease progression, even in the androgen ablated state, suggests PSA may have a role in the pathogenesis of disease. Current experimental and clinical evidence suggests chronic inflammation, irrespective of the cause, may predispose men to PCa. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated yet not completely understood. In this study, we used a mass spectrometric based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of immunoglobulins, as well as complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed not only the high-level expression of C3 in the prostatic fluid but also the presence of a previously uncharacterized Cterminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. Together these results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that may help to create an environment hospitable to malignancy through proteolysis of the complement system. INTRODUCTION Prostate-specific antigen (PSA) is a serine protease that is a unique differentiation product of prostate tissue. PSA is one of the most abundant proteins in the seminal plasma where it is present at mg/ml concentrations. While the exact physiologic role of PSA remains unknown, its major substrates in the seminal plasma are the gel-forming proteins semenogelin (Sg) I and II (1–3). PSA is able to maintain the seminal plasma in a semi-liquid state through cleavage of these gel-forming proteins. PSA is also produced in high amounts by prostate cancer cells. A role for PSA in the pathobiology of prostate cancer has been proposed based on its effect on prostate cancer growth (4) and its ability to cleave several important growth regulatory proteins (5). However, the exact role for PSA in prostate cancer has yet to be clearly defined. PSA is not expressed by any other tissue in the adult human male and leaks out of prostate cancer sites with disrupted tissue architecture. On this basis PSA has utility as a biomarker for prostate cancer. The overwhelming majority of men with prostate cancer, even those with poorly differentiated, high grade disease, continue to express PSA at high levels throughout the course of disease progression. The word prostate is derived from Greek and literally means “one who stands before” or “protector” (6). While the exact role of the prostate gland is not clear, it is the guardian of the genitourinary tract and prevents foreign materials from entering the reproductive apparatus of the male. In light of this role, the prostate of the aging male exhibits significant chronic inflammation that may lead to the development of prostate cancer (7). However, while the prostate tissue may be pro-inflammatory, the prostatic fluid is not, as evidenced by the fact that men with prostatitis commonly have no or minimal inflammatory cells in the prostatic secretions. Immunoregulation within the prostatic fluid must also be finely balanced. The fluid must have the capability to eliminate foreign bacteria and viruses entering the genitourinary tract through the urethra. It must also shield the sperm from immune destruction within the vaginal tract while at the same time not eliminating cells within the reproductive tract of the female. In this regard, seminal plasma is devoid of complement activity, and actually has a strong anti-complement activity (8–10). In this study, we used a mass spectrometric based evaluation of prostatic fluid obtained from cancercontaining prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of immunoglobulins, as well as complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed not only the high-level expression of C3 but also a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C-terminal cleavage fragment revealed a putative PSA cleavage site which was confirmed using purified PSA and C3. Further studies revealed PSA to preferentially cleave iC3b, itself a cleavage product resulting from complement activation. We then tested whether this activity had functional consequences on CR3 activation, but could not detect any. Finally we determined that the evolutionarily-related complement protein C5, but not C4, is a substrate of PSA as well. PSA-mediated proteolysis of C5 inhibits complement pathway activity. Together these results suggest a previously unknown function of PSA as an immunoregulatory protease that may help to create an environment hospitable to malignancy through inactivation of the complement system. Finally, these findings suggest PSA may also have immunoregulatory activity in the seminal plasma to aid in normal fertility that may have been co-opted by prostate cancer cells as a means to avoid immune destruction. MATERIALS AND METHODS Patient samples and cell lines. Prostatic fluid samples were collected from radical prostatectomy specimens as previously described according to an Institutional Review Board approved protocol (11). Seminal plasma was obtained from discarded clinical samples. The RAW 264.7 macrophage cell line was obtained from ATCC. Mass spectrometric sample preparation and analysis. Individual prostatic fluid samples were loaded into the wells of a 4-12% Bis-Tris NuPage gel. Following electrophoretic separation the gel was stained with SimplyBlue SafeStain (Invitrogen). Individual gel lanes were excised into 12 similar sized pieces and each piece was placed into a separate microcentrifuge tube. The gel slices were destained with water before being immersed into 500 μl of 100 mM ammonium bicarbonate. In-gel tryptic digestion was performed on all gel slices (1:20 ratio trypsin enzyme:substrate) for 18 hours at 37°C. Mass spectrometric analysis and subsequent protein identifications were performed as previously described (4). Western blot. Prostatic fluid samples stored at -80°C were thawed, centrifuged, and protein concentrations in the supernatant were determined by the BCA method. Proteins (5 μg) were separated by SDS-PAGE and then transferred to PVDF membrane (Bio-Rad). Membranes were blocked with 4% nonfat milk in TBS-Tween 0.1%. Primary and secondary antibodies were prepared in the same diluent. The membrane was probed with monoclonal anti-human-C3b-α (1:10,000; clone H206) from Millipore and ECL-anti-mouse IgG (1:8000) from GE Healthcare. The membrane was incubated with SuperSignal West Pico Substrate (Pierce) then exposed to X-ray film. Immunoaffinity purification. Polyclonal anti-C3 (Complement Technology) was covalently linked to AminoLink Coupling Resin (Pierce) by following the manufacturer’s instructions. Briefly, 16.5 mg antibody was diluted into 2 mL coupling buffer before adding 40 μL sodium cyanoborohybride. This mixture was added to 2 mL of resin and incubated for 5 hours under gentle agitation. The column was washed then remaining active sites were blocked before additional washing. Four prostatic fluid samples were pooled then diluted to 1.5 mL in TBS. Samples were added to the prepared column and binding occurred for 1 hour. The column was washed then elution buffer was added and 1 mL fractions were collected. Fractions containing relevant protein were concentrated using an Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane (Millipore). Edman degradation. Concentrated immunopurified prostatic fluid was separated on a 4-15% gel and transferred to a PVDF membrane. The membrane was cut in half where a small amount of immunopurified sample was probed with the anti-human-C3b-α antibody as described above. The remaining membrane was incubated with Coomassie stain before a brief destain. The X-ray film was overlaid onto the Coomassie-stained membrane to identify the correct band which was then excised and sent to the Johns Hopkins Synthesis and Sequencing Facility for Edman degradation. The first seven N-terminal amino acids were determined with a Perkin-Elmer/Applied Biosystems Procise Protein Sequencing System. Coincubation of C3/C3b/iC3b and PSA. Purified human C3, C3b, and iC3b (Complement Technology) were incubated with enzymatically active PSA (AbD Serotec) in the presence of 10 μM aprotinin (Sigma). PSA inhibitor (1 μM) was added to control reactions. Reactions took place in PSA buffer (50 mM Tris, 100 mM NaCl, pH = 7.5) overnight at 37 ̊C. Reactions were stopped by addition of sample loading buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membrane as described above. Membranes were stained with Coomassie blue, briefly destained, then digitally imaged. The band at ~37 kDa was excised and sent for Edman degradation as described above. Determination of cofactor activity. Purified human C3b was incubated with enzymatically active PSA and an increasing amount of factor H (Quidel). Reactions took place in PSA buffer overnight at 37 ̊C. Reactions were stopped by addition of sample loading buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membrane as described above. C3b/iC3b deposition assay. Sheep erythrocytes (ES) were opsonized with C3b as described (12). iC3b opsonized sheep erythrocytes were prepared by incubating antibody sensitized sheep erythrocytes (EA) with C5depleted serum. Approximately 2 million EA (Complement Technology) were mixed with 10 μL of normal human serum stripped of C5 by immunoaffinity chromatography (C5 (-) NHS) in triplicate. After 20 minutes at 37 ̊C erythrocytes were washed twice with PBS. ES and EA were resuspended in PSA or BSA (125 μg/mL) in the presence of aprotinin (10 μM) then incubated at 37 ̊C for 2 hours on a rotisserie mixer. Cells were washed once with PBS then resuspended in a 10 μg/mL solution of anti-human-C3b-α (clone H206) and incubated for 1 hour on ice. Cells were washed once with PBS then resuspended in a 10 μg/mL solution of anti-mouse IgG Alexa Fluor 488 and incubated for 30 minutes on ice in the dark. Cells were washed with PBS then fixed with formalin. Levels of C3b-α were measured by a BD FACSCalibur at the Sidney Kimmel Comprehensive Cancer Center Flow Cytometry Core Facility. CR3-mediated phagocytosis of EA-iC3b. Assessment of complement-mediated phagocytosis was performed as described (13, 14). EA-iC3b were prepared as described above. EA-iC3b were incubated with enzymatically active PSA or BSA in PSA buffer overnight at 37 ̊C. RAW 264.7 cells were propagated in DMEM supplemented with 10% FBS in a humidified atmosphere of 5% CO2 at 37°C. The cells were seeded on polylysine coated 96-well plates such that they were 90% confluent on the day of experimentation. RAW 264.7 cells were stimulated with 125 ng/μL phorbol 12-myristate 13-acetate (PMA, Promega) for 10 minutes at 37 ̊C. EAiC3b pretreated with PSA or BSA were added to the stimulated RAW 264.7 cells and phagocytosis proceeded for 75 minutes at 37°C. Phagocytosis was quantified colorimetrically by the conversion of 2,7-diaminofluorene by hemoglobin into a product that absorbs at 620 nm. The RAW 264.7 cells were washed twice in PBS. Erythrocytes that had bound but not been internalized were lysed by a brief incubation in 0.2% NaCl. The RAW 264.7 cells were again washed twice in PBS before being lysed with 50 μL of 6M urea in 0.2M Tris-HCl pH=7.4. The cell lysates were mixed with 75 μL of working solution (10 volumes 0.2M Tris-HCl pH=7.4, 1 volume 2,7-diaminofluorene stock, and 0.02 volumes 30% hydrogen peroxide). Absorbance at 620 nm was monitored with a plate reader. Coincubation of C4/C5 and PSA. Purified human C4 and C5 (Complement Technology) were incubated with enzymatically active PSA in the presence of 10 μM aprotinin in PSA buffer. After overnight incubation at 37 ̊C the reaction products were separated by SDS-PAGE and stained with SimplyBlue SafeStain. C5 supplementation of C5 (-) NHS. Purified human C5 was mixed with enzymatically active PSA or BSA and incubated overnight at 37 ̊C. The next day 50 μL of EA were supplemented with 2 μL of C5 (-) NHS. The C5 pretreated with PSA or BSA was added to the erythrocytes and incubated at 37 ̊C for 20 minutes. Reactions were centrifuged at 1000 x g and the supernatants were collected. The absorbance of the supernatant at 415nm was recorded. Comparison of C5 levels in serum, prostatic fluid, and seminal plasma. A Western blot was performed as described above. The membrane was probed with polyclonal anti-human-C5 (1:2,000) from Complement Technology and donkey anti-goat IgG-HRP (1:20,000) from Santa Cruz Biotechnology. Addition of C5 to fresh seminal plasma. Purified human C5 in PSA buffer was incubated with fresh seminal plasma for 2 hours at 37 ̊C. PSA inhibitor (10 μM) was added to control reactions. Reactions were stopped by addition of sample loading buffer. A Western blot was performed as described above. The membrane was probed with polyclonal anti-human-C5 (1:2,000) and donkey anti-goat IgG-HRP (1:20,000). RESULTS Mass spectrometric based identification of 95 proteins in prostatic fluid. All protein species identified from the prostatic fluid of each of 4 patients were introduced into the proteomic platform Protein Center (Thermo Fisher Scientific) as individual patient proteome files using the methodology outlined by Williams et al (4). A comparative analysis was performed to determine which proteins had been identified in all analyzed patient samples (Figure 1A and Supplemental Table I). The 95 proteins common to all 4 patients were introduced as an independent data set. Using the seminal plasma proteome published by Bartosz Pilch and Matthias Mann (15) as a reference database, the subset of common experimentally identified proteins was compared to the reference database. Of the 95 proteins included in the experimental data set, 58 had previously been identified in seminal plasma (Figure 1B). Both our dataset and the dataset of Pilch and Mann included proteins known to be expressed by the prostate at high levels such as PSA and prostatic acid phosphatase; inclusion of these proteins served as internal validation. Complement system proteins C3, factor B and clusterin were detected in all 4 patient samples. These three proteins were also present in the reference database. Additional complement proteins present in the reference database but not in our dataset included C1, C2, C4, C9, and complement factor I. C3 and a previously uncharacterized C3 fragment are present in diseased prostatic fluid and normal seminal plasma. To confirm the results from our proteomic study, we analyzed eight additional prostatic fluid samples from men with prostate cancer by Western blot to confirm the presence of C3. The antibody for this analysis, monoclonal anti-human-C3b-α (clone H206), is directed towards an epitope present on the α-chain of the C3 protein. While the exact epitope recognized by this antibody is not known, it is able to detect both C3b and C3c consistent with detection of an epitope towards the C-terminus of the C3 α-chain (16). C3 was detected in all eight prostatic fluid samples and in the seminal plasma of a healthy male (Figure 1C). While equal amounts of protein were loaded for each sample varying levels of C3 were detected by Western blot. Six of the eight prostatic fluid samples also tested positive for a C3 fragment of approximately 105 kDa, which most likely represented C3b, which is evidentiary of complement activation. All eight prostatic fluid samples and the seminal plasma from a healthy donor also tested positive for a 37 kDa fragment using the anti-C3b-α antibody. This 37 kDa fragment was not detected in the serum of healthy individuals or in the serum of patients with prostate cancer (data not shown). Of the previously described C3 cleavage fragments this 37 kDa fragment appears to be closest in size to C3c α-chain fragment 2. Characterization of the novel 37 kDa C3 fragment. C3 is a well characterized protein whose activation and degradation is tightly regulated. Following conversion to C3b by the C3 convertase complex, C3b is subsequently inactivated by the proteolytic activity of factor I in the presence of co-factor molecules factor H, CR1, or CD46/membrane co-factor protein (MCP). Factor I cleavage generates multiple previously characterized cleavage fragments that include C3c, C3dg and C3f (17) (Figure 2). To better characterize this putative C3 fragment, immunoaffinity purification was utilized to purify the 37 kDa fragment from prostatic fluid for further characterization. Purification was achieved using a polyclonal C3 antibody. Because prostatic fluid sample volume was limited, four samples were pooled prior to purification. Seven cycles of Edman degradation on the purified pooled prostatic fluid samples revealed the amino acid sequence of the N-terminus of the 37 kDa fragment to be “HAKAKDQ”. Comparison to the C3 reference sequence indicates the 37 kDa fragment is indeed a previously undescribed C3 fragment that maps to the 36.5 kDa C-terminal portion of the C3 α-chain (Figure 2). This 37 kDa fragment is detectable under reducing conditions by Western blot due to its release from the N-terminal portion of the C3 α-chain following reduction of the disulfide bond. Further inspection of the sequence flanking the cleavage site revealed that the new N-terminus was created by a chymotrypsin-like protease with cleavage after tyrosine-1348 in the C3 protein. In contrast, all other previously described C3 cleavage fragments are produced following cleavage by trypsin-like proteases. Furthermore, cleavage at tyrosine-1348 to generate the 37 kDa fragment, like every other previously described C3 fragment, is the result of cleavage within the C3 α-chain. C3 β-chain cleavage fragments have not been described. PSA can cleave C3 and generate the 37 kDa fragment in vitro. PSA is the major chymotrypsin-like serine protease in the seminal plasma and prostatic fluid. Therefore, we hypothesized that PSA was cleaving C3 based on sequence similarity between known PSA substrates and the cleavage sequence N-terminal to the tyrosine1348 within C3, “TLSVVTMY//HAKAKDQ” (Figure 2). To test this we incubated purified human C3 with purified enzymatically active PSA. Addition of a potent and specific PSA inhibitor (18) served as a negative control. Reducing gel electrophoresis revealed no significant cleavage of the C3 α-chain (Figure 3A). This led us to hypothesize tyrosine-1348 was part of a cryptic site exposed after proteolytic activation of C3 into C3b or iC3b. To test this we incubated purified human C3b and iC3b with purified enzymatically active PSA, again using a PSA inhibitor as a negative control. Reducing gel electrophoresis revealed degradation of both fragments of the iC3b α-chain, however no effect was observed with C3b (Figure 3A). Cleavage of iC3b resulted in a fragment at a similar molecular weight as that observed following similar analysis of the prostatic fluid samples. To confirm this cleavage product was the same proteolytic fragment detected in the prostatic fluid we excised and sequenced the 37 kDa band by Edman degradation. The N-terminus of the PSA generated fragment was confirmed to be “HAKAKDQ” consistent with cleavage after tyrosine-1348. Factor H does not have cofactor activity to facilitate PSA-mediated cleavage of C3b. Factor I is unable to cleave C3b in the absence of the cofactor Factor H. Therefore, we hypothesized that factor H may also have cofactor activity for PSA enabling it to cleave C3b. To test this we repeated our C3b proteolysis assay with PSA in the presence of complement factor H (Figure 3B). Results show factor H does not impart any cofactor activity on PSA to mediate cleavage of C3b. PSA cleaves iC3b, but not C3b, deposited on the surface of sheep erythrocytes. The next experiments were performed to determine whether PSA could cleave C3b or iC3b in a more relevant cellular context. Sheep erythrocytes (ES) were opsonized with C3b using purified C3 and alternative pathway enzymes factor B and factor D in the absence of factor I and H to prevent cleavage of C3b to iC3b. Antibody-sensitized sheep erythrocytes (EA) were opsonized with iC3b by brief incubation with C5-depleted normal human serum. The addition of C5-depleted serum ensures the complement activation pathway only proceeds through deposition of C3b on the cell membrane and prevents the formation of the membrane attack complex and subsequent cell lysis. Factors I and H present in the C5-depleted serum converts C3b into iC3b. ES-C3b and EA-iC3b were incubated with enzymatically active PSA at 37 ̊C. The erythrocytes were collected and labeled with monoclonal anti-human-C3b-α (clone H206) and analyzed by flow cytometry. Analysis revealed a decrease in C3b-α antibody signal when EA-iC3b were treated with 125 μg/mL PSA (approximately a 10-fold lower level of PSA than that observed in the prostatic fluid (19)) compared to the signal observed when cells were treated with the same concentration of BSA (Figure 4A) . Treatment of ES-C3b with PSA did not result in a decrease of the C3b-α antibody signal (Figure 4B). To determine whether PSA was releasing the 37 kDa iC3b fragment into the supernatant we collected and tested it for the presence of C3 fragments by Western blot. Two C3 fragments, one at 37 kDa and another at 39 kDa, were detected consistent with a combination of factor I and PSA cleavage (Figure 4C). PSA-mediated cleavage of EA-iC3b does not alter complement-dependent phagocytosis. After conversion of C3b to its inactivated form, iC3b can no longer bind factor B and act as a C3 convertase. However iC3b and its degradation product C3dg are active molecules which trigger specialized immune responses by interacting with complement receptors on leukocytes (20). Complement-dependent phagocytosis is an important mechanism of the host defense system and is primarily mediated by complement receptor CR3, and to a lesser extent CR1 and CR4. CR3 is expressed on many immune cells including macrophages, monocytes, and neutrophils. C3b does not interact with CR3, and iC3b is predicted to interact with CR3 through binding sites which become exposed upon unfolding of the CUB domain after cleavage of the C3b α-chain (21). We hypothesized that PSA’s ability to cleave iC3b between the CUB and MG8 domain on the α-chain might interfere with CR3 binding. To test this we used an established protocol to measure complement-dependent phagocytosis (14). In this assay the CR3 RAW 264.7 macrophage cell line internalizes iC3b opsonized sheep erythrocytes. PSA treated EA-iC3b were prepared as usual and were added to pre-stimulated RAW cells at a 20:1 ratio. Phagocytosis was stopped and bound cells that had not internalized were lysed by addition of a hypotonic solution. A sensitive colorimetric assay that relies on the pseudoperoxidase activity of hemoglobin was used to evaluate the phagocytic efficiency (13). Cells were lysed and hemoglobin was released from internalized EA-iC3b. The relative internalization can be measured by the pseudoperoxidase activity of hemoglobin which coverts 2,7-diaminofluorene into fluorene blue which can be measured spectrophotometrically. This sensitive method of detection did not demonstrate any difference in the degree of phagocytosis between PSA-treated and control EA-iC3b (Figure 5). PSA also cleaves the homologous C5 protein. The complement system is a collection of over 30 different proteins. Three key components (C3, C4, and C5) are thought to have evolved from a common ancestor, and all share a similar molecular weight and chain structure (22). Because of the similarities between the three proteins we were curious if C3 was uniquely cleaved by PSA or if all were substrates of PSA. We treated C4 and C5 with enzymatically active PSA and looked for cleavage products by electrophoresis. We could not detect any significant proteolysis of the C4 αor β-chains (Figure 6A). The α-chain of C5 exhibited significant proteolytic degradation, while the β-chain was left intact (Figure 6B), similar to what we observed with C3. PSA-mediated cleavage of C5 has functional consequences. We were curious if PSA-mediated cleavage of C5 had functional consequences on the integrity of the complement cascade. To test this we used EA to assay total complement hemolytic activity. C5 was incubated with PSA overnight. The following day we supplemented C5-depleted normal human serum with PSA-treated C5 or control C5, and added it to EA. We observed significantly less complement activity in the sample supplemented with PSA-treated C5 compared to control C5, indicating that PSA-mediated proteolysis of C5 negatively regulates the complement pathway (Figure 6C). Proteolysis of C5 in the seminal plasma can be abrogated by a PSA inhibitor. Seminal plasma is a rich source of proteins, including proteins of the complement system (15). However, unlike serum, this fluid is not a source of fully functional complement, likely due to both the presence of complement inhibitory proteins and the absence of certain complement factors. Notably missing in the seminal plasma is C5 (Figure 7A). We were curious if the lack of C5 in seminal plasma might be due in part to PSA proteolytic activity. To answer this question we supplemented seminal fluid with purified human C5 in the presence or absence of a PSA inhibitor. We then determined C5 levels by Western blot with a polyclonal antibody. In the absence of a PSA inhibitor seminal plasma was able to degrade the α-chain of C5, leaving the β-chain intact (Figure 7B). DISCUSSION Complement is regarded as one of the first lines of immunological defense, defending the host from foreign invaders by one of three pathways of activation known as the classical pathway, alternative pathway, and lectin pathway (23). Complement factor C3 plays a central role in the complement cascade and supports the activation of all three pathways. Human C3 is the most abundant complement protein in the serum and, based on our proteomic studies of the prostatic fluid, is also one of the most abundant proteins in the seminal plasma. C3 is highly regulated both prior to and following activation by C3 convertases. Cleavage by C3 convertases releases the anaphylatoxin C3a and generates C3b. Once formed, C3b rapidly attaches via covalent bond formation to various acceptors on the surface of bacteria and host cells. Since C3b does not have the ability to discriminate between self and non-self, it has the potential to damage host cells. Therefore, membrane bound C3b activity must be regulated by other complement proteins. In this regard C3b expresses multiple binding sites for other complement components that either amplify its convertase activity (factor B and properdin in the presence of factor D) or inactivate its activity (proteolysis by factor I in the presence of factor H, CR1 or CD46). C3b’s factor I mediated degradation product, iC3b, has an equally interesting biology. iC3b interacts with CR2, CR3, and CR4, the first of which plays a role in enhancing B-cell immunity. iC3b’s other receptor binding partners, CR3 and CR4, play a role in clearance of pathogens by phagocytosis. In this study we provide initial evidence that human PSA, via its chymotrypsin-like serine protease activity, can modulate the complement system through degradation of iC3b to produce new C3 degradation fragments and through degradation of the complement protein C5, thereby inactivating the complement cascade. In this study, PSA was shown to cleave iC3b and was unable to cleave C3 or C3b. C3 is known to undergo a significant conformational change upon activation into C3b and then again following deactivation into iC3b by sequential proteolysis (21, 24–27). High-resolution crystal structures exist for both C3 and C3b documenting these conformational changes. These crystal structures detect a conformational change of up to 95Å and the exposure of cryptic binding sites. Examination of the crystal structures of C3 and C3b (PDB ID 2A73 and 2I07, respectively) reveal that the PSA cleavage site at tyrosine-1348 is part of a β-strand facing the interior of the protein, making it an inaccessible substrate of PSA. Unfortunately, we have a limited understanding of the structure of iC3b. The conversion from C3b to iC3b likely results in significant shifts and the generation of cryptic binding sites much like the earlier conversion from C3 to C3b. iC3b, but not C3b, interacts with CR2, CR3, and CR4, so these sites must be hidden in C3b but made accessible upon conversion to iC3b. Low resolution 3D-electron microscopy analysis of iC3b confirms a significant conformational change upon conversion from C3b to iC3b, but cannot provide atomic resolution (26). Our results indicate only iC3b to be a substrate of PSA, suggesting conversion into iC3b makes tyrosine-1348 accessible to the solvent and thus PSA-mediated proteolysis. To confirm this proteolytic activity could be duplicated in a more relevant cellular context we repeated the assay with C3b and iC3b covalently attached to the surface of sheep erythrocytes (ES-iC3b and EA-iC3b, respectively) and analyzed it by flow cytometry. Following C3 activation, C3b becomes attached to cell membranes due to formation of a covalent bond between the C3b protein and the cell surface. This bond is formed when exposed hydroxyl and amine groups on cell surface proteins and carbohydrates interact with the reactive thioester bond within the C3b protein. C3b is subject to factor I proteolysis resulting in iC3b, itself an important protein which uniquely interacts with CR2, CR3, and CR4. We treated both EA-iC3b and ES-C3b with PSA, but observed a decrease in antibody signal only with EA-iC3b, consistent with removal of part or all of iC3b from the erythrocyte surface. After PSA treatment the 37 kDa iC3b fragment could be detected in the supernatant. PSA-mediated cleavage of iC3b after tyrosine-1348 alone would not liberate the 37 kDa fragment from the surface of the cell due to disulfide bonds linking the iC3b α-chain fragments 1 and 2. Electrophoretic analysis indicates additional PSA-mediated cleavage of iC3b (Figure 3A), including cleavage of the α-chain fragment 1, which would release the 37 kDa fragment from surface of the erythrocyte. Unfortunately cleavage of the α-chain fragment 1 appears to be nearly complete, making characterization of these cleavage fragments technically challenging. In the absence of PSA the α-chain fragment 2 (39.5 kDa) can also be detected in the supernatant (Figure 4C), indicating additional proteolysis is occurring, perhaps by factor I and the appropriate