Bitiscetin-3, a Novel C-Type Lectin-like Protein Cloned from the Venom Gland of the Viper Bitis arietans, Induces Platelet Agglutination and Inhibits Binding of Von Willebrand Factor to Collagen.

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and-at least bitiscetin-1-induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein-bitiscetin-3-were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and ß share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3ß were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit-which differs from that of both bitiscetin-3 subunits-the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and ß-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

Strategy for generation of replication-competent recombinant rotaviruses expressing multiple foreign genes.

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

Effects of plasma glycosyltransferase on the ABO(H) blood group antigens of human von Willebrand factor.

Von Willebrand factor (VWF) is one of the plasma protein carrying ABO(H) blood group antigens, but the combining process of these antigens is not clear. In the present study, we examined whether plasma glycosyltransferase affects the blood group antigens on VWF. VWF expressing H-antigen (H-VWF) from blood group O and bovine serum albumin conjugated with H-antigen (H-BSA) were incubated with recombinant α1-3-N-acetylgalactosaminyltransferase (rA-transferase) and A-plasma with or without an additional UDP-GalNAc. Transformed antigens were detected by western blotting and ELISA, using an anti-A antibody. Both H-VWF and H-BSA acquired the A-antigen after incubation with rA-transferase and UDP-GalNAc. Incubation with A-plasma very weakly converted the H-antigen on BSA and VWF to A-antigen only in the presence of supplemented UDP-GalNAc. This conversion was enhanced on desialylation of H-VWF. These results indicate that sugar chains of plasma VWF can be modified by the external glycosyltransferase, but that plasma glycosyltransferase has no effect on the blood group antigens of VWF due to its low activity and the lack of donor sugars. Further, sialic acid residues of VWF may exert a protective effect against post-translational glycosylation. Our results clearly exclude the possibility that blood group antigens of VWF are constructed extracellularly in plasma.

Spatiotemporal expression pattern of Myt/NZF family zinc finger transcription factors during mouse nervous system development.

BACKGROUND: Three members of the Myt/NZF family of transcription factors are involved in many processes of vertebrate development. Several studies have reported that Myt1/NZF-2 has a regulatory function in the development of cultured oligodendrocyte progenitors or in neuronal differentiation during Xenopus primary neurogenesis. However, little is known about the proper function of Myt/NZF family proteins during mammalian nervous system development. To assess the possible function of Myt/NZF transcription factors in mammalian neuronal differentiation, we determined the comparative spatial and temporal expression patterns of all three types of Myt/NZF family genes in the embryonic mouse nervous system using quantitative reverse transcriptase polymerase chain reaction and in situ hybridization. RESULTS: All three Myt/NZF family genes were extensively expressed in developing mouse nervous tissues, and their expression was transient. NZF-1 was expressed later in post-mitotic neurons. NZF-2 was initially expressed in neuronal cells a little earlier than NZF-3. NZF-3 was initially expressed in neuronal cells, just after proliferation was complete. CONCLUSION: These expression patterns suggest that the expression of NZF family genes is spatially and temporally regulated, and each Myt/NZF family gene may have a regulatory function in a specific phase during neuronal differentiation.

Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination.

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the ß subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the ß subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the ß subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspß70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the ß subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

Myt/NZF family transcription factors regulate neuronal differentiation of P19 cells.

During mammalian central nervous system development, neural stem cells differentiate and then mature into various types of neurons. Myelin transcription factor (Myt)/neural zinc finger (NZF) family proteins were first identified as myelin proteolipid protein promoter binding factors and were shown to be involved in oligodendrocyte development. In this study, we found that Myt/NZF family molecules were expressed during neuronal differentiation in vivo and in vitro. Transient over-expression of Myt/NZF family genes could convert undifferentiated P19 cells into neurons without induction by retinoic acid (RA), and the ability of these genes to induce neuronal differentiation was comparable to that of Neurog1 and Neurod1. Additionally, we found that St18 (or NZF-3) was induced by several bHLH transcription factors. When NZF-3 and Neurog1 were co-expressed in P19 cells, the rate of neuronal differentiation was significantly increased. These data suggest not only that NZF-3 works downstream of Neurog1 but also that it plays a crucial role together with Neurog1 in neuronal differentiation.

NZF-2b is a novel predominant form of mouse NZF-2/MyT1, expressed in differentiated neurons especially at higher levels in newly generated ones.

NZF-2 (MyT1) is a member of C2HC-type zinc finger transcription factors. A novel form of mouse NZF-2 has been isolated. This novel form, NZF-2b, has an additional C2HC-type zinc finger motif. The expression levels of NZF-2b are by far the more predominant than those of the already known form of NZF-2. In embryonic mouse nervous system, the expression of NZF-2b starts as early as at 9.5 days post-coitum (dpc) in newly differentiated neurons in the central nervous system (CNS) and the peripheral nervous system (PNS).