Protein substratesProtein substrate cleavage sequence Fibrinogen α-chainGPR Fibrinogen β-chainSAR Factor XIII A-chainVPR C2LGR C3LAR Relative catalytic activity Tripeptide substrate sequence 1FGR 0.107VPR 0.106DPR 0.064IEGR 0.05FSR 0.043QGR 0.020GGR 0.010VLK 0.007LGR Table 5.1. The catalytic activity of MASP1 against tripeptide-AMC substrates. The catalytic activity is listed relative to FGR-AMC, since it is the one for which MASP1 has the highest catalytic activity. The data are compiled from Presanis et al., (2004) Table 5.2. Protein cleavage sequences for MASP1. The data were compiled from Matsushita et al., (2000) (C2 and C3), Hajela et al., (2002) (fibrinogen α-chain), while the remaining is from this thesis.

rMASP1 reduced rMASP1 non-reduced A B Figure 5.1. Panel A shows a schematic representation of full-length MASP1 and the truncated rMASP1. The difference between the two is that the rMASP1 lacks the three N-terminal domains which are involved in the complex formation with MBL and the ficolins. The arrows indicate the Arg 448 -Ile 449 activation site that is cleaved upon activation. Panel B shows rMASP1 when run non-reduced and reduced on SDS-PAGE. Non-reduced at 47 kDa band can be seen, while two bands at 30 kDa and 20 kDa band corresponding to the serine protease domain and the truncated heavy chain appear when run reduced. For a detailed description of the experimental setup see section SP-domain CCP 1 CCP 2

Hours 1 Hour 2 Hours4 Hours 8 Hours SP domain CCP 1 CCP 2 AB Figure 5.2. Stability of rMASP1. In panel A rMASP1 was incubated for 0, 1, 2, 4 and 8 hours at 37 o C and the loss of activity was estimated by measuring VPR-AMC turnover. The %-activity is compared to the sample incubated 0 hours at 37 o C. After 1 hour 20% loss of activity is observed while only 35% is lost at 8 hours. In panel B the autolysis of rMASP1 can be observed when incubated at the same time intervals. The 30 kDa and the 20 kDa bands are the SP domain and the CCP 1 CCP 2 domains, respectively. After 1 hour of incubation a third band appears, which is a degradation product of the SP-domain. This band increases in intensity upon incubation and less than 20% SP-domain remains after 8 hours. The fragment lost from the SP-domain can also be observed at approximately 6 kDa after 4 hours of incubation. Both of the degradation products are indicated by unlabelled arrows. A higher molecular weight band is seen at 8 hours and may be an artifact, but corresponds approximately to a SP domain dimer. For a detailed description of the experimental setup see section

Figure 5.3. The NaCl and Ca 2+ ion dependence of rMASP1 in 20 mM HEPES, pH 7.4 buffer. When rMASP1 activity (cleavage of VPR-AMC) was measured at different NaCl concentrations the activity is highest at low salt strengths. This is true when incubated in the presence of 5 mM Ca 2+ or EDTA but in the presence of EDTA the activity is approximately 15% lower overall than when incubated with Ca 2+ ions. The % of max activity is calculated based on the rMASP1 activity at 16 mM NaCl, 5 mM Ca 2+, taken as 100%. For a detailed description of the experimental setup see section

Plasma IgG depleted plasma IgG and HAS depleted plasma IgG heavy chain IgG light chain Figure 5.4. SDS-PAGE analysis of 80 μg protein from plasma, IgG-depleted plasma and HSA- and IgG-depleted plasma. In the non-depleted plasma four major bands can be seen. The bands indicated by the arrows are IgG heavy and light chains and as can be observed they are diminished >90% after IgG depletion. The HSA (66 kDa) is very dominant in the plasma and the IgG-depleted plasma but the intensity of the band is greatly reduced after depletion. This shows that the plasma has been depleted sufficiently for IgG and HSA. For a detailed description of the experimental setup see section

Figure 5.5. Gel filtration of HSA- and IgG-depleted plasma. Panel A shows the OD 280 trace from the Superose 12 column. V 0 is the void volume and V t indicates the total volume. Panel B, SDS-PAGE of the fractions following the void volume. For a detailed description of the experimental setup see section ml32-34 ml ml ml38-40 ml40-42 ml ml ml46-48 ml48-50 ml50-52 ml52-54 ml54-56 ml56-58 ml58-60 ml60-88 ml A B ml

mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl mM NaCl - Figure 5.6. SDS-PAGE analysis of MonoQ fractions of the Superose ml pool with or without digestion of rMASP1. mM NaCl indicates at what ionic strength the proteins were eluted from the MonoQ column. Addition of rMASP1 is indicated by + and if no rMASP1 was added a -. In the mM NaCl fraction the two bands that differ between the rMASP1 treated and non-treated lane have been marked by arrows. The first (1) is a novel band not present at all in the untreated sample while the second (2) is found in both lanes but the intensity of it is increased several fold when rMASP1 is present. The 2 nd band is also found at increased intensity in the and mM NaCl fractions when treated with rMASP1. The three lower unmarked arrows indicate the positions of bands originating from rMASP1 alone. For a detailed description of the experimental setup see section

NH 3 treated + NH 3 treated - Non-treated + Non-treated - Figure 5.7. The effect of rMASP1 on an ammonia-treated compared to a non-treated sample. Panel A: The sample analyzed is the fraction eluting at mM NaCl from the MonoQ column (figure 5.6). The samples to which were added rMASP1 are labeled (+) while those without are labeled (-). The two bands of interest (>212 kD and 85 kDa) are both found in the non-treated sample while in the ammonia- treated sample the >212 kDa band has disappeared and the 85 kDa is greatly diminished. Since only proteins with thiolester bonds like C4, C3 and α2M are sensitive to ammonia treatment this indicates that the formation of the two bands most likely is due to the action of rMASP1 on α2M. The arrow points to the band, which underwent N-terminal sequencing. The result from the N-terminal sequencing shows that the first 10 amino acids were SVSGKPQYMV, which identifies the band as being α2M. For a detailed description of the experimental setup see section NH 3 treated + NH 3 treated - Non-treated + Non-treated Sequence S V S G K P Q Y M V AB

SVSGKPQYMV LVPSLLHTET TEKGCVLLSY LNETVTVSAS LESVRGNRSL FTDLEAENDV LHCVAFAVPK SSSNEEVMFL TVQVKGPTQE FKKRTTVMVK NEDSLVFVQT DKSIYKPGQT VKFRVVSMDE NFHPLNELIP LVYIQDPKGN RIAQWQSFQL EGGLKQFSFP LSSEPFQGSY KVVVQKKSGG RTEHPFTVEE FVLPKFEVQV TVPKIITILE EEMNVSVCGL YTYGKPVPGH VTVSICRKYS DASDCHGEDS QAFCEKFSGQ LNSHGCFYQQ VKTKVFQLKR KEYEMKLHTE AQIQEEGTVV ELTGRQSSEI TRTITKLSFV KVDSHFRQGI PFFGQVRLVD GKGVPIPNKV IFIRGNEANY YSNATTDEHG LVQFSINTTN VMGTSLTVRV NYKDRSPCYG YQWVSEEHEE AHHTAYLVFS PSKSFVHLEP MSHELPCGHT QTVQAHYILN GGTLLGLKKL SFYYLIMAKG GIVRTGTHGL LVKQEDMKGH FSISIPVKSD IAPVARLLIY AVLPTGDVIG DSAKYDVENC LANKVDLSFS PSQSLPASHA HLRVTAAPQS VCALRAVDQS VLLMKPDAEL SASSVYNLLP EKDLTGFPGP LNDQDDEDCI NRHNVYINGI TYTPVSSTNE KDMYSFLEDM GLKAFTNSKI RKPKMCPQLQ QYEMHGPEGL RVGFYESDVM GRGHARLVHV EEPHTETVRK YFPETWIWDL VVVNSAGVAE VGVTVPDTIT EWKAGAFCLS EDAGLGISST ASLRAFQPFF VELTMPYSVI RGEAFTLKAT VLNYLPKCIR VSVQLEASPA FLAVPVEKEQ APHCICANGR QTVSWAVTPK SLGNVNFTVS AEALESQELC GTEVPSVPEH GRKDTVIKPL LVEPEGLEKE TTFNSLLCPS GGEVSEELSL KLPPNVVEES ARASVSVLGD ILGSAMQNTQ NLLQMPYGCG EQNMVLFAPN IYVLDYLNET QQLTPEVKSK AIGYLNTGYQ RQLNYKHYDG SYSTFGERYG RNQGNTWLTA FVLKTFAQAR AYIFIDEAHI TQALIWLSQR QKDNGCFRSS GSLLNNAIKG GVEDEVTLSA YITIALLEIP LTVTHPVVRN ALFCLESAWK TAQEGDHGSH VYTKALLAYA FALAGNQDKR KEVLKSLNEE AVKKDNSVHW ERPQKPKAPV GHFYEPQAPS AEVEMTSYVL LAYLTAQPAP TSEDLTSATN IVKWITKQQN AQGGFSSTQD TVVALHALSK YGAATFTRTG KAAQVTIQSS GTFSSKFQVD NNNRLLLQQV SLPELPGEYS MKVTGEGCVY LQTSLKYNIL PEKEEFPFAL GVQTLPQTCD EPKAHTSFQI SLSVSYTGSR SASNMAIVDV KMVSGFIPLK PTVKMLERSN HVSRTEVSSN HVLIYLDKVS NQTLSLFFTV LQDVPVRDLK PAIVKVYDYY ETDEFAIAEY NAPCSKDLGN A Figure 5.8. The amino acid sequence of the α2M subunit (SWISS-Prot entry: P01023) without the signal peptide. The sequence in red is the bait region which contains target sequences for many different proteases.

Figure 5.9. Factor XIII activation by thrombin and rMASP1. In panel A factor XIII was mixed with 1 U/ml thrombin and incubated for 0, 0.5, 1 2, 4, 8, 16 and 32 minutes. At 0 minutes only the factor XIII A- and B-chains are detectable but as time progresses more and more A-chain is cleaved generating the A’-chain. In panel B factor XIII was incubated with 10 μg/ml rMASP1 for 0, 5, 10, 20, 40, 80, 160 and 320 minutes. As with thrombin the A’-chain is generated as time progresses. For a detailed description of the experimental setup see section A B 5 min 10 min 20 min 40 min 80 min 160 min 0 min320 min 0 min 0.5 min 1 min 2 min 4 min 8 min 16 min32 min A chain A’ chain B chain A chain A’ chain B chain

A BC Factor XIII alone Factor XIII + rMASP1 SETSRTAFGG RRAVPPNNSN AAEDDLPTVE LQGVVPRGVN LQEFLNVTSV HLFKERWDTN KVDHHTDKYE NNKLIVRRGQ SFYVQIDFSR PYDPRRDLFR VEYVIGRYPQ ENKGTYIPVP IVSELQSGKW GAKIVMREDR SVRLSIQSSP KCIVGKFRMY VAVWTPYGVL RTSRNPETDT YILFNPWCED DAVYLDNEKE REEYVLNDIG VIFYGEVNDI KTRSWSYGQF EDGILDTCLY VMDRAQMDLS GRGNPIKVSR VGSAMVNAKD DEGVLVGSWD NIYAYGVPPS AWTGSVDILL EYRSSENPVR YGQCWVFAGV FNTFLRCLGI PARIVTNYFS AHDNDANLQM DIFLEEDGNV NSKLTKDSVW NYHCWNEAWM TRPDLPVGFG GWQAVDSTPQ ENSDGMYRCG PASVQAIKHG HVCFQFDAPF VFAEVNSDLI YITAKKDGTH VVENVDATHI GKLIVTKQIG GDGMMDITDT YKFQEGQEEE RLALETALMY GAKKPLNTEG VMKSRSNVDM DFEVENAVLG KDFKLSITFR NNSHNRYTIT AYLSANITFY TGVPKAEFKK ETFDVTLEPL SFKKEAVLIQ AGEYMGQLLE QASLHFFVTA RINETRDVLA KQKSTVLTIP EIIIKVRGTQ VVGSDMTVTV QFTNPLKETL RNVWVHLDGP GVTRPMKKMF REIRPNSTVQ WEEVCRPWVS GHRKLIASMS SDSLRHVYGE LDVQIQRRPS M Sequence G V N L Q Figure Cleavage of factor XIII by rMASP1. Panel A shows the amino acid sequence of the factor XIII A-chain (SWISS-Prot entry: P00488) without the signal peptide. The sequence outlined in red is the activation peptide that is removed after cleavage by thrombin. Panel B shows factor XIII incubated alone and with 32 μg/ml rMASP1 for 5 hours at 37 o C. The arrow indicates the band that was sequenced and panel C shows the initial 5 amino acids of the indicated band.

μg rMASP1 2 μg rMASP1 1 μg rMASP1 0.5 μg rMASP1 4 μg Thrombin 2 μg Thrombin 1 μg Thrombin 0.5 μg Thrombin Figure Purity and quantification of the amount of active enzyme in the rMASP1 and thrombin preparations. rMASP1 (4, 2, 1 and 0.5 μg) and thrombin (4, 2, 1 and 0.5 μg) were analyzed on SDS-PAGE to approximate the percentage of active protease to total protein in the rMASP1 and thrombin preparations. In the rMASP1 preparation the SP domain is a prominent band and there is no sign of degradation products. Similarly for the thrombin preparation, the SP domain is a clearly defined band, and it appear that both the rMASP1 and thrombin preparations are in a form which should be active. For a detailed description of the experimental setup see section Thrombin SP domain rMASP1 SP domain

μg/ml rMASP1 1 μg/ml rMASP12 μg/ml rMASP14 μg/ml rMASP1 8 μg/ml rMASP1 16 μg/ml rMASP132 μg/ml rMASP1 γ 2 -chain β-chain α-chain γ-chain γ 2 -chain β-chain α-chain γ-chain 10 U/ml Thrombin 5 U/ml Thrombin 2.5 U/ml Thrombin 1.25 U/ml Thrombin 0.63 U/ml Thrombin 0.32 U/ml Thrombin 0.16 U/ml Thrombin 0 U/ml Thrombin A B Figure Fibrinogen cleavage by thrombin and rMASP1 when incubated 24 hours at 37 o C. In panel A fibrinogen was incubated with a 2-fold dilution series of thrombin. A control sample without thrombin was also included. In the control sample 3 major bands can be observed, the α-, β- and γ-chains. In all the thrombin treated samples only two bands are visible, indicating that the reaction has reached its end point. The activated β-chain and the γ 2 -chain while the α-chain is not seen. However a product likely to be α n -chain is seen in the wells, and another minor product possibly an α-chain oligomer, is seen above the 158 kDa marker. When fibrinogen is treated with rMASP1 (panel B) a similar pattern emerges. The γ 2 -chain gradually appears while the α-chain gradually is diminished but the reaction does not reach the endpoint for the γ 2 formation. For a detailed description of the experimental setup see section α n -chain

QGVNDNEEGF FSARGHRPLD KKREEAPSLR PAPPPISGGG YRARPAKAAA TQKKVERKAP DAGGCLHADP DLGVLCPTGC QLQEALLQQE RPIRNSVDEL NNNVEAVSQT SSSSFQYMYL LKDLWQKRQK QVKDNENVVN EYSSELEKHQ LYIDETVNSN IPTNLRVLRS ILENLRSKIQ KLESDVSAQM EYCRTPCTVS CNIPVVSGKE CEEIIRKGGE TSEMYLIQPD SSVKPYRVYC DMNTENGGWT VIQNRQDGSV DFGRKWDPYK QGFGNVATNT DGKNYCGLPG EYWLGNDKIS QLTRMGPTEL LIEMEDWKGD KVKAHYGGFT VQNEANKYQI SVNKYRGTAG NALMDGASQL MGENRTMTIH NGMFFSTYDR DNDGWLTSDP RKQCSKEDGG GWWYNRCHAA NPNGRYYWGG QYTWDMAKHG TDDGVVWMNW KGSWYSMRKM SMKIRPFFPQ Q A C B Fibrinogen alone Fibrinogen + rMASP1 Figure Panel A shows The primary amino acid sequences of the fibrinogen β- chain without the signal peptide (SWISS-Prot entry: P02675). The sequence in red is the amino acids within fibrinopeptide B. Panel B shows fibrinogen incubated with or without 32 μg/ml rMASP1 for 16 hours at 37 o C and the arrow indicate the band that underwent N-terminal sequencing. Panel C shows the 6 amino acids (GHRPLD) that the new N-terminus is composed of. This shows that the rMASP1 and thrombin have the same cleavage site on the fibrinogen β-chain suggesting that both enzymes release fibrinopeptide B Sequence G H R P L D

μg/ml rMASP1 1 μg/ml rMASP1 2 μg/ml rMASP1 4 μg/ml rMASP18 μg/ml rMASP1 16 μg/ml rMASP132 μg/ml rMASP1 5 U/ml Thrombin 2.5 U/ml Thrombin 1.25 U/ml Thrombin 0.6 U/ml Thrombin 10 U/ml Thrombin 0 U/ml Thrombin 0.3 U/ml Thrombin 0.15 U/ml Thrombin A B β-chain α-chain γ-chain β-chain α-chain γ-chain Figure Fibrinogen cleavage by dilution series of thrombin (A) and rMASP1 (B) in the presence of IAM. When fibrinogen is incubated with low concentrations of thrombin the α- and β-chains shift down due to removal of the fibrinopeptides A and B. At higher concentrations of thrombin the α-chain is degraded into at least two smaller species that can be observed just above the β-chain. The γ-chain band broadens at the highest concentrations of thrombin indicating either that proteolysis occurs or more likely that α-chain degradation fragments co-runs with it. When fibrinogen is treated with rMASP1 the α-chain is degraded and gradually disappears but the degradation pattern is different from that seen with thrombin. The β-chain shifts down due to release of fibrinopeptide B while the γ-chain band broadens probably due to α-chain degradation products. The degradation products are more easily observed below 47 kDa. The unlabeled arrows in panel B indicated the bands believed to originate from the rMASP1 preparation. For a detailed description of the experimental setup see section

Peak no Predominant Peptide Molecular Weight (Da) Identity Fibrinopeptide A Fibrinopeptide B 3No peptide detected No peptide detected 10No peptide detected 11No peptide detected 12No peptide detected Fibrinopeptide A Fibrinopeptide B Fibrinopeptide A 19No peptide detected Fibrinopeptide B Figure Detection of fibrinopeptide A and B. In panel A the HPLC elution profiles of the peptides generated during fibrinogen incubation with thrombin and rMASP1 are shown. From the top panel down the samples are as follows: Fibrinopeptide A and B in a 4:1 (w/w) ratio with thrombin; fibrinogen alone; fibrinogen incubated with rMASP1; fibrinogen incubated for 16 hours with thrombin and fibrinogen incubated for 5 min with thrombin, respectively. Notice the different scales on the Y-axis and the numbering of the observed peaks. The red dotted line indicates the elution point for fibrinopeptide A and the blue the same for fibrinopeptide B. In panel B the molecular masses (Da) of the predominant peptide species in the numbered peaks is shown. The elution point and molecular mass is used for identification of the fibrinopeptides A and B. As can be observed in panel A both thrombin and rMASP1 cleave at multiple sites when incubated overnight but rMASP1 does seem to have more secondary cleavage sites than thrombin does and when incubated with rMASP1 neither fibrinopeptides A nor B could be detected. When thrombin incubation for 16 hours is compared to 5 min a clear difference can be observed since the fibrinopeptide B peak is much more predominant in the latter. For a detailed description of the experimental setup see section A B min Fibrinopeptide A and B + thrombin Fibrinogen alone Fibrinogen + rMASP1 Fibrinogen + thrombin (16 hours) Fibrinogen + thrombin (5 min) mAU 215

Figure Detection of fibrinopeptide A and B. The HPLC elution profiles from samples in which fibrinogen was incubated with thrombin or rMASP1 for 16 hours with or without 1 mM IAM. The red and blue dotted lines indicate the elution points for fibrinopeptides A and B, respectively. Notice the change of scale on the Y-axis. As can be observed does the incubation with IAM not change the profiles i.e. make the fibrinopeptide B peak appear neither in the case of thrombin nor rMASP1. Thus it is unlikely that the disappearance of fibrinopeptide B is due to factor XIIIa mediated cross-linking. Due to this the loss of fibrinopeptide B is more likely to be due to secondary degradation. For a detailed description of the experimental setup see section Thrombin + fibrinogen Thrombin + fibrinogen + IAM rMASP1 + fibrinogen + IAM rMASP1 + fibrinogen min mAU 215