von Willebrand factor A1 domain can adequately substitute for A3 domain in recruitment of flowing platelets to collagen.

BACKGROUND: Binding of von Willebrand factor (VWF) to platelet GPIbalpha and to collagen is attributed to VWF A1 and A3 domains, respectively. OBJECTIVES: Using VWF, VWF lacking A1 (DeltaA1-VWF) or A3 (DeltaA3-VWF) and VWF with defective A3 (H1786A-VWF), in combination with recombinant A1 (residues 1262-1492) or A3 (residues 1671-1878), fused to glutathione-S-transferase (GST-A1 and GST-A3), we have re-investigated the role of A1 in platelet recruitment to surfaces of collagen. METHODS AND RESULTS: In flow, measurable binding of DeltaA3-VWF occurred to horse tendon, but also to human type III collagen. GST-A1 and GST-A3 both competed for binding of DeltaA1-VWF and DeltaA3-VWF to horse tendon collagen fibrils in static conditions and to human collagen III during plasmon surface resonance studies, substantiating overlapping binding sites on both collagens for A1 and A3. Heparin did not affect A3-mediated binding of VWF and DeltaA1-VWF, but inhibited binding to horse tendon collagen of GST-A1 and DeltaA3-VWF. Furthermore, A1-mediated binding to type III collagen of DeltaA3-VWF binding was strongly salt-sensitive. During perfusions at wall shear rate 2500 s(-1) of calcein-labeled platelets in reconstituted blood, DeltaA3-VWF and H1786A-VWF triggered platelet binding to horse tendon collagen comparably and as potently as VWF, and to human type III collagen, only fivefold less potently, DeltaA1-VWF being inactive. Additional flow-controlled interaction studies with DeltaA3-VWF, H1786A-VWF, the collagen-VWF antagonist saratin, heparin and the VWF neutralizing antibody 82D6A3 confirmed that H1786A-VWF binds to collagen exclusively via A1. CONCLUSION: Hence, in shear forces the VWF A1 domain can assume the role of A3 to trigger substantial platelet recruitment to human collagen fibres.

Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries.

The Tn611 transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons. The resulting plasmid, pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis. These are the first mycobacterial insertional mutant libraries to be constructed by transposition directly into a mycobacterium. No highly preferential insertion sites were detected by Southern blot analysis of the chromosomal DNAs isolated from the insertion mutants. Auxotrophic mutants with various phenotypes were isolated at a frequency ranging from 0.1 to 0.4%, suggesting that the libraries are representative. The pCG79 system thus seems to be a useful tool for the study of M. smegmatis genetics and may be applicable to other mycobacteria, such as the M. tuberculosis complex.

Molecular cloning, characterization and purification of ornithine carbamoyltransferase from Mycobacterium bovis BCG.

A genomic library of Mycobacterium bovis BCG has been constructed by cloning DNA partially digested with Sau3A into the Escherichia coli expression vector pAS1. The gene coding for ornithine carbamoyl-transferase (EC.2.1.3.3; OTCase), hereafter referred to as argF, was isolated from the library by complementation of a double argF-argI mutant of E. coli and its sequence was determined. The translation initiation codon used, GTG, was identified by comparing the amino acid sequence deduced from the gene with the N-terminal sequence of the corresponding purified protein. On this basis, the M. bovis BCG OTCase monomer consists of 307 amino acid residues and displays about 44% identity with other OTCases, the most closely related homologue being the anabolic enzyme of Pseudomonas aeruginosa. The native enzyme has an estimated molecular mass of 110 kDa, suggesting a trimeric structure as is the case for most of the anabolic OTCases known from various organisms.