Yu-Liang Xiong

Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys.

We transplanted kidneys from α1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy.

Background. The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. Methods. Seven baboons received life-supporting kidney transplants from hDAF transgenic pigs. Anti-Gal antibodies were neutralized by GAS914 or TPC (a Gal PEG glycoconjugate polymer). Group 1 (n=5) underwent a conventional immunosuppressive therapy with FK506, rabbit anti-thymocyte serum/immunoglobulin, mycophenolate mofetil, and steroids. Group 2 (n=2) received an anti-humoral immunity regimen with LF15-0195, Rituxan and cobra venom factor in addition to ATG, FK506 and steroids. Levels of anti-non-Gal antibodies and their mediated complement-dependent cytotoxic activities (CDC) were detected by flow cytometry using Gal knockout (k/o) pig lymphocytes (LC) or endothelial cells (EC) as targets. Results. Continuous infusion of GAS914/TPC significantly reduced anti-Gal antibodies. In Group 1, four of five baboons developed severe acute humoral xenograft rejection (AHXR) and the rejection was associated with either a high level of preformed anti-non-Gal IgG or a marked elevation in induced anti-non-Gal IgG and IgM. Sera collected at the time of AHXR had a high level of CDC to porcine LC/EC from Gal k/o animals. The intensive anti-humoral therapy in Group 2 completely inhibited both anti-Gal and non-Gal antibody production and prevented AHXR. However, this therapy was not well tolerated by the baboons. Conclusion. In a pig-to-baboon kidney transplant model, both preformed and induced anti-non-Gal antibodies are strongly associated with the pathogenesis of AHXR when anti-Gal antibodies are neutralized.

Complement inhibition enables renal allograft accommodation and long-term engraftment in presensitized nonhuman primates.

Protection against humoral injury mediated by donor‐specific antibodies (DSA), also known as accommodation, may allow for long‐term allograft survival in presensitized recipients. In the present study, we determined the role of complement in renal allograft accommodation in donor skin‐presensitized nonhuman primates under conventional immunosuppression. Donor skin allografts were transplanted to presensitized recipients 14 days prior to renal transplantation. Renal allografts not receiving any immunosuppressive treatment developed accelerated rejection with predominantly humoral injury, which was not prevented using conventional cyclosporine (CsA) triple therapy. Inhibition of complement activation with the Yunnan‐cobra venom factor (Y‐CVF) successfully prevented accelerated antibody‐mediated rejection and resulted in successful accommodation and long‐term renal allograft survival in most presensitized recipients. Accommodation in this model was associated with the prevention of the early antibody responses induced against donor antigens by complement inhibition. Some antiapoptotic proteins and complement regulatory proteins, including Bcl‐2, CD59, CD46 and clusterin, were upregulated in the surviving renal allografts. These results suggest that the complement inhibition‐based strategy may be valuable alternative in future clinical cross‐match positive or ABO‐incompatible transplantation.

Purification and characterization of a new L-amino acid oxidase from Daboia russellii siamensis venom.

A new L-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom L-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic L-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates--L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 microg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 microM) and TMVA (55.0 nM) with an IC(50) value of 32.8 microg/ml and 32.3 microg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 microg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 microg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 microg/ml.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejnegeri) venom

From the venom of Chinese green tree viper (Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-1, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatography (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mol. wt of 50000, 31000 and 32000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnefibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA and stejnobin, stejnefibrase-1, -2 and -3 did not activate plasminogen and did not possess fibrinogen-clotting activity. The three purified enzymes directly degraded fibrinogen to small fragments and rendered it unclottable by thrombin. Stejnefibrase-2 degraded preferentially Bbeta-chain while stejnefibrase-1 and -3 cleaved concomitantly Aalpha and Bbeta-chains of fibrinogen. None of these proteases degraded the gamma-chain of fibrinogen. When correlated with the loss of clottability of fibrinogen, the most active enzyme was stejnefibrase-1. The activities of the three enzymes were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB), indicating that like TSV-PA and stejnobin, they are venom serine proteases.

An activator of blood coagulation factor X from the venom of Bungarus fasciatus

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom.

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15536 Da) and beta (14873 Da). The mature amino acid sequences of the alpha (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers of (alpha beta)(2) and (alpha beta)(4) with molecular weights of 63680 and 128518 Da, respectively. The (alpha beta)(2) complex is stabilized by an interchain disulfide bridge between the two alpha beta-heterodimers, whereas the stabilization of the (alpha beta)(4) complex seems to involve non-covalent interactions between the (alpha beta)(2) complexes.

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

Trimeresurus stejnegeri venom which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin.

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii.

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin, was purified by DEAE A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH(2)-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg. The fibrinopeptides released, identified by HPLC, consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it, indicating it is venom serine protease.